ABCMdb

ABCC7 p.Gly1349Asp

Admin's notes:	Class III (gating defect) Veit et al.
ClinVar:	c.4046G>A , p.Gly1349Asp D , Pathogenic c.4045G>A , p.Gly1349Ser ? , not provided
CF databases:	c.4046G>A , p.Gly1349Asp D , CF-causing ; CFTR1: We tested 20 non-[delta]F508 CF chromosomes and did not find a second example of this mutation. The mutation destroys an NcoI site. c.4045G>A , p.Gly1349Ser (CFTR1) D , The mutation was found using SSCP analysis and direct sequencing. It was detected in one of the CFTR alleles of a Japanese CBAVD patient. The patient has another mutation Q1352H in the other allele.
Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (66%), E: D (95%), F: D (95%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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III (gating defect) <a href="https://www.ncbi.nlm.nih.gov/pubmed/26823392">Veit et al.</a>
admin on 2016-08-19 15:16:22

Publications
[hide] Novel pharmacologic therapies for cystic fibrosis. J Clin Invest. 1999 Feb;103(4):447-52. Zeitlin PL
Novel pharmacologic therapies for cystic fibrosis.
J Clin Invest. 1999 Feb;103(4):447-52., [PMID:10021451]

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125 These mutants sustain a reduced response to ATP-examples include S1255P, G551S, G1244E, and G1349D. Login to comment

[hide] Pharmacologic restoration of delta F508 CFTR-media... Kidney Int. 2000 Mar;57(3):832-7. Zeitlin PL
Pharmacologic restoration of delta F508 CFTR-mediated chloride current.
Kidney Int. 2000 Mar;57(3):832-7., [PMID:10720936]

Abstract [show]
Cystic fibrosis (CF) is an autosomal inherited disorder caused by over 800 different mutations in the CFTR gene. The most common mutation, delta F508, causes a trafficking arrest in the endoplasmic reticulum and the CFTR protein is degraded. Restoration of CFTR trafficking in vitro restores cAMP-mediated chloride transport at the cell surface. The hypothesis of this discussion is that the short chain fatty acids, butyrate and 4-phenylbutyrate, up-regulate mature CFTR at the plasma membrane. Evidence that these compounds regulate CFTR production and maturation in part through effects on molecular chaperones in CF cells in culture is discussed. The oral drug, 4-phenylbutyrate, was tested in a Phase I clinical trial in CF subjects and further trials are underway. Other new therapeutic approaches directed at different classes of mutations in CFTR are also discussed. Chemical and pharmacologic agents that regulate endogenous gene expression at different steps in the biosynthetic processing pathway of a membrane glycoprotein will be needed to comprehensively treat a complex inherited disorder like cystic fibrosis.

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98 These mutants sus- ment of CF cells with 4-phenylbutyrate or low tempera- tain a reduced response to adenosine 5Ј-triphosphate ture to induce ⌬F508 trafficking to the plasma mem- (ATP); examples include S1255P, G551S, G1244E, and brane, allowed genistein to activate chloride transport G1349D. Login to comment

[hide] A novel mutation in the CFTR gene correlates with ... J Med Genet. 2000 Mar;37(3):215-8. Wang J, Bowman MC, Hsu E, Wertz K, Wong LJ
A novel mutation in the CFTR gene correlates with severe clinical phenotype in seven Hispanic patients.
J Med Genet. 2000 Mar;37(3):215-8., [PMID:10777364]

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320 Since ATP hydrolysis at NBD2 terminates a burst of activities associated with opening the channel, loss of NBD2 would confer a loss of the gating control.21 A recent study shows that the Walker A motif in NBD2 is more solvent accessible than that in NBD1, suggesting a diVerence in structure and function for the two NBDs.22 In addition to 3876delA, a few other mutations in NBD2, including G1244E, S1255P, S1255X, 3905insT, W1282X, N1303K, and G1349D, all result in a PI phenotype.5 23 It should be noted that S1255P, S1255X, 3905insT, and 3876delA are all clustered around the Walker A motif. Login to comment

[hide] Type I, II, III, IV, and V cystic fibrosis transme... Curr Opin Pulm Med. 2000 Nov;6(6):521-9. Choo-Kang LR, Zeitlin PL
Type I, II, III, IV, and V cystic fibrosis transmembrane conductance regulator defects and opportunities for therapy.
Curr Opin Pulm Med. 2000 Nov;6(6):521-9., [PMID:11100963]

Abstract [show]
Recent advances in cellular and molecular biology have furthered the understanding of several genetic diseases, including cystic fibrosis. Mutations that cause cystic fibrosis are now understood in terms of the specific molecular consequences to the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and function. This knowledge has spawned interest in the development of therapies aimed directly at correcting the defective CFTR itself. In this article, we review the molecular defect underlying each recognized class of CFTR mutation and the potential therapies currently under investigation. Opportunities for protein-repair therapy appear to be vast and range from naturally occurring compounds, such as isoflavonoids, to pharmaceuticals already in clinical use, including aminoglycoside antibiotics, butyrate analogues, phosphodiesterase inhibitors, and adenosine nucleotides. Future therapies may resemble designer compounds like benzo[c]quinoliziniums or take the form of small peptide replacements. Given the heterogeneity and progressive nature of cystic fibrosis, however, optimal benefit from protein-repair therapy will most likely require the initiation of combined therapies early in the course of disease to avoid irreparable organ damage.

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92 These mutants, including S1255P, G551S, G1244E, and G1349D, sustain a reduced response to ATP. Login to comment
116 Through this mechanism, adenosine indirectly activates wild-type as well as several surface-localized mutant CFTR channels including R117H, A455E, and G1349D. Login to comment

[hide] Aberrant CFTR-dependent HCO3- transport in mutatio... Nature. 2001 Mar 1;410(6824):94-7. Choi JY, Muallem D, Kiselyov K, Lee MG, Thomas PJ, Muallem S
Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis.
Nature. 2001 Mar 1;410(6824):94-7., 2001-03-01 [PMID:11242048]

Abstract [show]
Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl(-)-coupled HCO3- transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO3- and pH affect mucin viscosity and bacterial binding. We have examined Cl(-)-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.

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36 Similar rates were measured for the I148T, G178R, A1067T, G1244E, S1255P and G1349D mutants (see Fig. 3 for location of these mutations in CFTR), all of which are associated with CF with pancreatic insuf®- ciency. Login to comment
186 letters to nature 96 NATURE |VOL 410 |1 MARCH 2001 |www.nature.com HCO3 -/Cl- transportratio 0 0.25 0.50 0.75 1.00 WT I148T G178R R297Q G551D H620Q G970R A1067T G1244E S1255P G1349D E193K G551S A800G H949Y R1070Q Pancreatic insufficient Pancreatic sufficientD648V N CI148T G178R E193K R297Q R117H A1067T R1070Q G1244E S1255P G1349D NBD2 RD H949Y G970R CL4CL3CL2CL1 NBD1 G551D G551S H620Q D648V A800G Figure 3 The HCO3:Cl-transport ratio of CFTR mutants associated with CF. Login to comment

[hide] Genotype-phenotype correlation in cystic fibrosis:... Am J Med Genet. 2002 Jul 22;111(1):88-95. Salvatore F, Scudiero O, Castaldo G
Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes.
Am J Med Genet. 2002 Jul 22;111(1):88-95., 2002-07-22 [PMID:12124743]

Abstract [show]
More than 1,000 mutations have been identified in the cystic fibrosis (CF) transmembrane regulator (CFTR) disease gene. The impact of these mutations on the protein and the wide spectrum of CF phenotypes prompted a series of Genotype-Phenotype correlation studies. The CFTR genotype is invariably correlated with pancreatic status-in about 85% of cases with pancreatic insufficiency and in about 15% of cases with pancreatic sufficiency. The correlations between the CFTR genotype and pulmonary, liver, and gastrointestinal expression are debatable. The heterogeneous phenotype in CF patients bearing the same genotype or homozygotes for nonsense mutations implicated environmental and/or genetic factors in the disease. However, the discordant phenotype observed in CF siblings argued against a major role of environmental factors and suggested that genes other than CFTR modulate the CF phenotype. A locus that modulates gastrointestinal expression was identified in mice and subsequently in humans. By analyzing nine CF patients discordant for meconium ileus we were able to show that this locus had a dominant effect. Moreover, in a collaborative study we found a higher rate of polymorphisms in beta-defensin genes 1 and 2 in CF patients and in controls. In another multicenter study mutations in alpha-1 antitrypsin (A1AT) and mannose binding lectin genes were found to be independent risk factors for liver disease in CF patients. The body of evidence available suggests that the variegated CF phenotype results from complex interactions between numerous gene products.

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46 A series of mutations usually associated with pancreatic sufficiency have been identified and defined as ''mild`` with reference to pancreatic status [Kerem et al., 1989c]: G85E, G91R, R117H, E193K, P205S, R334W, T338I, R347H, R347L, R347P, R352Q, A455E, S492F, S549N, P574H, D579G, 711 þ 5 G > A, C866Y, F1052V, H1054D, R1066H, R1068H, H1085R, D1152H, S1159P, S1251N, F1286S, G1349D, 2789 þ 5 G > A, and 3849 þ 10kb C > T [Dean et al., 1990; Cutting et al., 1990a; Cremonesi et al., 1992; Highsmith et al., 1994]. Login to comment

[hide] Down-regulation of volume-sensitive Cl- channels b... Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7. Ando-Akatsuka Y, Abdullaev IF, Lee EL, Okada Y, Sabirov RZ
Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain.
Pflugers Arch. 2002 Nov;445(2):177-86. Epub 2002 Sep 7., [PMID:12457238]

Abstract [show]
Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

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5 In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Login to comment
94 Crucial role of NBD2 in the CFTR-VSOR interaction To clarify the molecular basis for the CFTR-VSOR interaction, we first introduced mutations into NBD1 (G551D), NBD2 (G1349D) and the C-terminal PDZ-binding domain (DTRL). Login to comment
98 Quantitative densitometry (Fig. 4Bb) revealed that the G551D, G1349D and DTRL mutants were expressed to a comparable extent in the plasma membrane. Login to comment
116 Gly551 fiAsp and Gly1349 fiAsp (G551D and G1349D) are naturally occurring mutations in NBD1 and NBD2, known to cause a severe CF phenotype by decreasing nucleotide binding at NBDs [25]. Login to comment
120 In contrast to the mutation in NBD1, we found that the G1349D mutation failed to affect VSOR currents (Fig. 5), although it was expressed in the plasma membrane to an extent comparable to that of the G551D and DTRL mutants (Fig. 4). Login to comment
127 Fig. 5A, B Effects of expression of WT CFTR and CFTR proteins mutated at NBD1, NBD2 or the PDZ-binding domain on VSOR current densities in HEK293T cells. A Time-course of VSOR current activation by hypotonic stimulation of cells transfected with DTRL (top), G551D (middle) and G1349D (bottom) mutants, taken during application of alternating pulses from 0 to €40 mV every 15 s. B Peak VSOR current densities from HEK293T cells transfected with vector alone (Mock), WT CFTR or one of the three mutants, recorded at +40 mV after reaching a steady-state level. Login to comment
137 Since VSOR-non-regulating NBD2 mutants (G1349D, K1250M and D1370N) were expressed to approximately the same level as VSOR-regulating WT and G551D CFTR (as seen from immunostaining and Western blotting data), we may exclude the possibility that the down-regulation is simply a side-effect of overexpression of a foreign protein. Login to comment
171 On the contrary, the mutation G1349D in NBD2, which causes a severe CF phenotype with pancreatic insufficiency, effectively impaired the CFTR-VSOR interaction. Login to comment

[hide] Emerging drug treatments for cystic fibrosis. Expert Opin Emerg Drugs. 2003 Nov;8(2):523-35. Zeitlin PL
Emerging drug treatments for cystic fibrosis.
Expert Opin Emerg Drugs. 2003 Nov;8(2):523-35., [PMID:14662004]

Abstract [show]
Cystic fibrosis (CF) is one of the most common life-shortening inherited disorders. Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene disrupt the localisation and function of the cAMP-mediated chloride channel. Most of the morbidity and mortality arise from the lung disease which is characterised by excessive inflammation and chronic infection. Research into the mechanisms of wild-type and mutant CFTR biogenesis suggest that multiple drug targets can be identified. This review explores the current understanding of the nature of the different mutant CFTR forms and the potential for repair of the chloride channel defect. High-throughput screening, pharmacogenomics and proteomics bring recent technological advances to the field.

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88 Class of mutation Molecular mechanism Pancreatic status (if known) Examples 1 No CFTR protein synthesis PI W1282X, G542X, R553X, 621 + 1 G→T, 1717-1 G→A, 3905insT, 394delTT 2 Abnormal CFTR processing and trafficking PI ∆F508, N1303K, P574H 3 Defective CFTR regulation (normal trafficking) PI G551D, G551S, G1349D, S1255P 4 Decreased CFTR chloride conductance PS R117H, R334W, R347P, P547H 5 Reduced synthesis and trafficking of normal CFTR PS A455E, 3849 + 10kb C→T, (5T) 6A Reduced apical stability PI S1455X, Q1412S, 4326delTC, 4279insA 6B Defective regulation of other ion channels PI G551D Note that the G551D is placed in Class 3 for defective regulation and Class 6B for defective regulation of the outwardly rectifying chloride channel. Login to comment

[hide] Comprehensive cystic fibrosis mutation epidemiolog... Ann Hum Genet. 2005 Jan;69(Pt 1):15-24. Castaldo G, Polizzi A, Tomaiuolo R, Cazeneuve C, Girodon E, Santostasi T, Salvatore D, Raia V, Rigillo N, Goossens M, Salvatore F
Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italian population.
Ann Hum Genet. 2005 Jan;69(Pt 1):15-24., [PMID:15638824]

Abstract [show]
We screened the whole coding region of the cystic fibrosis transmembrane regulator (CFTR) gene in 371 unrelated cystic fibrosis (CF) patients from three regions of southern Italy. Forty-three mutations detected 91.5% of CF mutated chromosomes by denaturing gradient gel electrophoresis analysis, and three intragenic CFTR polymorphisms predicted a myriad of rare mutations in uncharacterized CF chromosomes. Twelve mutations are peculiar to CF chromosomes from southern Italy: R1158X, 4016insT, L1065P and 711 + 1G > T are present in 6.3% of CF chromosomes in Campania; G1244E and 852del22 are present in 9.6% of CF chromosomes in Basilicata and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D are frequent in Puglia (19.6% of CF alleles). Several mutations frequently found in northern Italy (e.g., R1162X, 711 + 5G > T) and northern Europe (e.g., G551D, I507del and 621 + 1G > T) are absent from the studied population. The I148T-3195del6 complex allele was present in two CF chromosomes, whereas I148T was present in both alleles (as a single mutation) in another CF patient and in five CF carriers; this could result from crossover events. The haplotype analysis of three intragenic polymorphisms (IVS8CA, IVS17bTA and IVS17bCA) compared with data from other studies revealed that several mutations (3849 + 10kbC > T, 1717-1G > A, E585X, 3272-26G > A, L558S, 2184insA and R347P) originated from multiple events, whereas others (R1158X and S549R) could be associated with one or more intragenic recombinant events. Given the large population migration from southern Italy, knowledge of the CF molecular epidemiology in this area is an important contribution to diagnosis, counselling and interlaboratory quality control for molecular laboratories worldwide.

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2 Twelve mutations are peculiar to CF chromosomes from southern Italy: R1158X, 4016insT, L1065P and 711+1G>T are present in 6.3% of CF chromosomes in Campania; G1244E and 852del22 are present in 9.6% of CF chromosomes in Basilicata and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D are frequent in Puglia (19.6% of CF alleles). Login to comment
49 In particular, 4016insT, R1158X, 711+1 G>T and L1065P had a cumulative frequency of 6.3% in CF chromosomes from Campania; G1244E and 852del22 a cumulative frequency of 9.6% in CF chromosomes from Basilicata; and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D a cumulative frequency of 19.6% in CF chromosomes from Puglia. Login to comment
62 A procedure for the large-scale analysis of several mutations peculiar to southern Italy is also indicated Mutation Analytical CF alleles Campania Basilicata Puglia Total procedure n = 340 n = 52 n = 350 n = 742 DF508 55.6 55.8 46.8 51.5 N1303K 7.3 3.8 7.7 7.3 G542X 5.0 3.8 7.1 5.9 W1282X 3.5 3.8 0.6 2.2 2183 AA>G 2.3 5.8 0.8 1.9 852del22 0 5.8 3.2 1.9 3% agarose 1717-1G>A 2.3 1.9 1.1 1.8 4382delA 0 0 3.7 1.8 RE (Ear I -) 1259insA 0 0 3.1 1.5 4016insT 2.1 0 1.1 1.5 ASO R553X 1.5 0 1.7 1.5 R1158X 1.5 0 1.3 1.2 ASO or RE (Sfa N 1 -) L1077P 0.6 0 1.9 1.2 I502T 0.3 0 2.0 1.1 RE (Mse I -) 3849+10kbC>T 0 1.9 1.6 0.9 D579G 0 0 1.6 0.8 RE (Avr II +) G1244E 0.9 3.8 0.3 0.8 ASO or RE (Mbo II +) G1349D 0 0 1.7 0.8 RE (Sty I -) 2789+5 G>A 0.6 0 0.8 0.7 711+1 G>T 1.5 0 0 0.7 ASO L1065P 1.2 0 0 0.5 ASO or RE (Mnl I +) R347P 0.3 0 0.9 0.5 2522insC 0.9 0 0 0.4 E585X 0.6 0 0 0.3 G85E 0.6 0 0 0.3 G178R 0.6 0 0 0.3 D1152H 0.3 0 0.3 0.3 I148T-3195del6 0.6 0 0 0.3 I148T (alone) 0 0 0.3 0.1 R334W 0 0 0.3 0.1 DI507 0 0 0.3 0.1 I1005R 0 0 0.3 0.1 3272-26A>G 0.3 0 0 0.1 2711delT 0.3 0 0 0.1 L558S 0 1.9 0 0.1 W1063X 0 0 0.3 0.1 D110H 0.3 0 0 0.1 S549R (A>C) 0 1.9 0 0.1 2184insA 0.3 0 0 0.1 3131del22 0.3 0 0 0.1 R709N 0 0 0.3 0.1 A349V 0 0 0.3 0.1 4015insA 0 0 0.3 0.1 Y849X 0 1.9 0 0.1 Cumulative 91.6 92.1 91.7 91.5 Unknown 8.4 7.9 8.3 8.5 Total 100,0 100,0 100,0 100,0 RE: restriction enzyme (-/+: abolition or introduction of a RE site); ASO: allele specific oligonucleotide Figure 2 Multiplex denaturing gradient gel electrophoretic analysis of exons 8, 5 and 18 of the cystic fibrosis transmembrane regulator gene in a cystic fibrosis patient (case n. Login to comment
86 However, 12 mutations (4016insT, R1158X, 711+1G>T, L1065P, G1244E, 4382delA, 1259insA, I502T, 852del22, D579G, L1077P and G1349D) have not been found (or have a low incidence) in populations from the British Isles (Cheadle et al. 1993; Ferec et al. 1992), Spain (Chillon et al. 1994; Casals et al. Login to comment
97 Due to the presence of 'local` mutations, the detection rate with commercial kits for CF chromosomes in Table 3 Mutations linked to different haplotypes possibly due to slippage events, characteryzed at the level of three CFTR intragenic loci (IVS8CA, IVS17bTA, IVS17bCA) by the indication of the repeats number Present study Other studies Cases Haplotype cases (n) (n. of repeats) (n) Haplotype references* (n. of repeats) R347P 4/4 16-32-13 3 16-32-13 1,2,3 1 16-31-13 3 2 17-28-13 1 1 16-45-13 1 L1077P 3/3 17-7-17 1 17-7-17 1 1 17-7-16 1 G85E 2/2 16-24-13 9 16-24-13 2,3 1 16-25-13 2 2183AA>G 14/14 16-31-13 1 16-31-13 3 4 16-30-13 1 R553X 6/11 17-55-13 3 17-58-13 3 3/11 18-55-13 1 17-57-11 1 1/11 16-55-13 2 17-55-13 1,3 1/11 16-55-11 6 17-55-11 1 1 17-52-11 1 1 17-54-11 1 1 17-56-13 3 G1244E 5/6 16-32-13 1 17-34-13 1 1/6 16-34-13 711 +1 G>T 5/5 16-25-13 7 16-25-13 1,2,3 1 16-26-13 1 G1349D 5/6 16-30-13 1/6 16-32-13 G178R 1/2 16-32-13 1 16-30-13 3 1/2 16-32-13 2 16-32-13 1 * References 1: Morral et al. 1996. Login to comment

[hide] Molecular pathology of the CFTR locus in male infe... Reprod Biomed Online. 2005 Jan;10(1):14-41. Claustres M
Molecular pathology of the CFTR locus in male infertility.
Reprod Biomed Online. 2005 Jan;10(1):14-41., [PMID:15705292]

Abstract [show]
Congenital bilateral absence of the vas deferens (CBAVD) is a form of infertility with an autosomal recessive genetic background in otherwise healthy males. CBAVD is caused by cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations on both alleles in approximately 80% of cases. Striking CFTR genotypic differences are observed in cystic fibrosis (CF) and in CBAVD. The 5T allele is a CBAVD mutation with incomplete penetrance. Recent evidence confirmed that a second polymorphic locus exists and is a major CFTR modifier. The development of minigene models have led to results suggesting that CFTR exon 9 is skipped in humans because of unusual suboptimal 5' splice sites. An extremely rare T3 allele has been reported and it has recently been confirmed that the T3 allele dramatically increases exon 9 skipping and should be considered as a 'CF' mutation. Routine testing for the most prevalent mutations in the CF Caucasian population will miss most CFTR gene alterations, which can be detected only through exhaustive scanning of CFTR sequences. Finally, a higher than expected frequency of CFTR mutations and/or polymorphisms is now found in a growing number of monosymptomatic disorders, which creates a dilemma for setting nosologic boundaries between CF and diseases related to CFTR.

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170 mutations so far attributed to this group are located within the NBD, such as missense mutations G55ID in NBDI or G1349D in NBD2. Login to comment

[hide] Binding site of activators of the cystic fibrosis ... Cell Mol Life Sci. 2005 Feb;62(4):446-60. Moran O, Galietta LJ, Zegarra-Moran O
Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains.
Cell Mol Life Sci. 2005 Feb;62(4):446-60., [PMID:15719171]

Abstract [show]
The use of substances that could activate the defective chloride channels of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) has been suggested as possible therapy for cystic fibrosis. Using epithelia formed by cells stably transfected with wildtype or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, K(D), of a series of CFTR activators by measuring the increase in the apical membrane current. Modification of apparent K(D) of CFTR activators by mutations of the nucleotide-binding domains (NBDs) suggests that the binding site might be in these regions. The human NBD structure was predicted by homology with murine NBD1. An NBD1-NBD2 complex was constructed by overlying monomers to a bacterial ABC transporter NBD dimer in the "head-to-tail" conformation. Binding sites for CFTR activators were predicted by molecular docking. Comparison of theoretical binding free energy estimated in the model to free energy estimated from the apparent dissociation constants, K(D), resulted in a remarkably good correlation coefficient for one of the putative binding sites, located in the interface between NBD1 and NBD2.

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2 Using epithelia formed by cells stably transfected with wild-type or mutant (G551D, G1349D) CFTR, we estimated the apparent dissociation constant, KD, of a series of CFTR activators by measuring the increase in the apical membrane current. Login to comment
32 Materials and methods Cell cultures Fisher rat thyroid (FRT) cells expressing WT, G551D or G1349D CFTR were cultured on 60-mm petri dishes with Coon`s modified F12 containing 5% fetal bovine serum, 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and 600 µg/ml zeocin, as previously described [18]. Login to comment
63 Compound WT G551D G1349D Apigenin(a) 3. Login to comment
64 1 ± 0.6 (4) 19.6 ± 6.1 (3) - Genistein(a) 19.6 ± 2.5 (15) 114.2 ± 12 (3) 34.38 ± 2.21 (3) UCCF-023(b) 0.5 ± 0.2 (3) - - UCCF-029(b) 1.5 ± 0.5 (4) 31.3 ± 3.5 (3) 11.7 ± 3.1 (5) UCCF-030(b) 0.5 ± 0.1 (3) 4.7 ± 1.3 (2) - UCCF-853(b) 1.17 (1) - - C02(b) 3.2 ± 0.3 (2) - - C03(b) 1.2 (1) 7.2 ± 3 (2) - Act01(c) 0.4 ± 0.04 (5) - 0.6 ± 0.2 (3) Act03(c) 0.07 ± 0.02 (2) - - Act04(c) 0.4 ± 0.08 (4) not active up to 20 µM - Act05(c) 0.13 ± 0.01 (4) not active up to 5 µM - Act09(d) 1.95 ± 0.7 (3) not active up to 50 µM - Act11(d) 0.3 ± 0.08 (3) not active up to 20 µM - Act12(d) 1.9 ± 1.3 (3) - - Act13(d) 0.2 ± 0.03 (3) not active up to 20 µM - Act14(d) 0.2 ± 0.04 (2) - - Act17(d) 1.1 ± 0.5 (6) - - Data were obtained from measurements of Isc on FRT cell monolayers expressing WT or mutant (G551D or G1349D) CFTR. Login to comment
84 Results Electrophysiology We compared the effect of different CFTR activators on the WT and on mutations of the signature sequences of CFTR, the NBD1 mutant G551D, and the mirror NBD2 mutant G1349D. Login to comment
91 We found that genistein also caused an Isc increase on the G1349D mutant (fig. 3A). Login to comment
92 The dose-response curve for the G1349D mutant was shifted to the right with respect to WT cells, but to a lesser extent than that of G551D (fig. 3B). Login to comment
94 In all cases, the affinities for the G551D mutant were significantly shifted to the right with respect to the WT protein, and the affinities for G1349D were in-between (fig. 3C, D). Login to comment
114 Effect of different CFTR activators on polarized epithelial preparations expressing WT or CFTR mutants (G1349D and G551D). Login to comment
117 Note that the lowest concentration that inhibited CFTR currents was 100 µM for WT and 200 µM for G1349D, while 200 µM still stimulated G551D. Login to comment
118 (B-D) Normalized dose-response relationships of FRT cells expressing WT (closed circles), G1349D (open triangles) and G551D (open squares) CFTR to genistein (B), UCCF-029 (C), and apigenin (D). Login to comment
170 The two mutations studied, G551D and G1349D, are located in the LSGGQ signature of NBD1 and NBD2, respectively. Login to comment
177 Hydrophobic (DGHB) and electrostatic (DGelec) contributions to interaction between NBD1 and NBD2 from WT and the mutants G551D and G1349D. Login to comment
178 WT G551D G1349D NBD1 + NBD2 ∆GHB (kJ/mol) -66.1 -68.4 -69.3 ∆Gelec (kJ/mol) -17.2 -12.5 -13.1 NBD1 + NBD2 + 2 ATP ∆GHB (kJ/mol) -98.4 -98.44 -101.05 ∆Gelec (kJ/mol) -28.9 -23.3 -24.3 Data were calculated from the interaction of NBDs in the absence of ATP, NBD1-NBD2, and from NBDs interacting in the presence of ATP, NBD1-NBD2-2 ATP. Login to comment
180 The hydrophobic contribution to the interaction energy is slightly improved in the mutants (DGHB = -68.4 kJ/mol for G551D, and DGHB = -69.3 kJ/mol for G1349D), but electrostatic terms of energy are significantly increased (DGElec = -12.5 kJ/mol for mutant G551D, and DGElec = -13.1 kJ/mol for mutant G1349D), with a net increase in the interaction energy of 2.9 kJ/mol and 1.4 kJ/mol for G551D and G1349D, respectively (table 2). Login to comment
184 Conversely, for mutant G1349D, the affinity forATP is reduced in site 1 (DDGbind = 11.1 kJ/mol), with a smaller effect on site 2 (DDGbind = 3.3 kJ/mol). Login to comment
205 Comparison of the binding free energy differences, DGbind estimated for ATP-binding sites 1 and 2, for WT and the mutants G551D and G1349D of human CFTR. Login to comment
206 WT WT-G551D WT-G1349D DDGbind (kJ/mol) DDGbind (kJ/mol) Site 1 1.83 11.06 Site 2 12.98 3.3 Site1-site 2 -2.78 13.92 -4.99 Changes are expressed as differences in DGbind (DDGbind), calculated at different conditions. Login to comment
242 Filled symbols represent data obtained docking the CFTR-activator to the WT model, open triangles are from G551D, and open circles are from G1349D. Login to comment
260 Now, we extend these observation to five substances tested on mutant G551D and three tested on mutant G1349D. Login to comment
261 Interestingly, there is a decrease of affinity for every CFTR activator tested on the mutants, the reduction being more marked for mutant G551D than for mutant G1349D (table 1). Login to comment
293 Similarly, as site 1 is more directly affected by mutation G1349D, a more severe reduction in ATP affinity is observed than in site 2. Login to comment
294 These results are in agreement with direct measurements of ATP binding done in NBDs of multidrug resistance protein-1 [31], where mutations G771D and G1433D of LSGGQ signatures, equivalent to G551D and G1349D in CFTR NBDs, produced similar effects on ATP-NBD interactions. Login to comment

[hide] Phenylglycine and sulfonamide correctors of defect... Mol Pharmacol. 2005 May;67(5):1797-807. Epub 2005 Feb 18. Pedemonte N, Sonawane ND, Taddei A, Hu J, Zegarra-Moran O, Suen YF, Robins LI, Dicus CW, Willenbring D, Nantz MH, Kurth MJ, Galietta LJ, Verkman AS
Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating.
Mol Pharmacol. 2005 May;67(5):1797-807. Epub 2005 Feb 18., [PMID:15722457]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel cause cystic fibrosis. The delta F508 mutation produces defects in channel gating and cellular processing, whereas the G551D mutation produces primarily a gating defect. To identify correctors of gating, 50,000 diverse small molecules were screened at 2.5 microM (with forskolin, 20 microM) by an iodide uptake assay in epithelial cells coexpressing delta F508-CFTR and a fluorescent halide indicator (yellow fluorescent protein-H148Q/I152L) after delta F508-CFTR rescue by 24-h culture at 27 degrees C. Secondary analysis and testing of >1000 structural analogs yielded two novel classes of correctors of defective delta F508-CFTR gating ("potentiators") with nanomolar potency that were active in human delta F508 and G551D cells. The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylace tamide, reversibly activated delta F508-CFTR in the presence of forskolin with K(a) approximately 70 nM and also activated the CFTR gating mutants G551D and G1349D with K(a) values of approximately 1100 and 40 nM, respectively. The most potent sulfonamide, 6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide, had K(a) approximately 20 nM for activation of delta F508-CFTR. In cell-attached patch-clamp experiments, phenylglycine-01 (PG-01) and sulfonamide-01 (SF-01) increased channel open probability >5-fold by the reduction of interburst closed time. An interesting property of these compounds was their ability to act in synergy with cAMP agonists. Microsome metabolism studies and rat pharmacokinetic analysis suggested significantly more rapid metabolism of PG-01 than SF-03. Phenylglycine and sulfonamide compounds may be useful for monotherapy of cystic fibrosis caused by gating mutants and possibly for a subset of delta F508 subjects with significant delta F508-CFTR plasma-membrane expression.

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6 The most potent compound of the phenylglycine class, 2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopro- pylphenyl)-2-phenylacetamide, reversibly activated ⌬F508-CFTR in the presence of forskolin with Ka ϳ 70 nM and also activated the CFTR gating mutants G551D and G1349D with Ka values of ϳ1100 and 40 nM, respectively. Login to comment
188 Activation of G551Dand G1349D-CFTR mutants. Login to comment
189 A and B, stimulation of apical membrane Cl- current by genistein (top) and PG-01 (bottom) in G551Dand G1349D-CFTR-expressing FRT cells. Login to comment
191 C and D, dose-responses for the PG-01 and genistein for activation of G551Dand G1349D-CFTR (S.E., n ϭ 4). Login to comment
217 Measurements were done in the "class III" mutants G551D and G1349D, which produce a severe gating defect without impairment in protein trafficking (Gregory et al., 1991). Login to comment
225 The G551D and G1349D mutant CFTRs produced little Cl- current after the addition of maximal forskolin (Fig. 6, A and B). Login to comment
226 Genistein, a known activator of G551Dand G1349D-CFTR, increased Cl- cur- rent substantially, albeit at high micromolar concentrations (Fig. 6, A and B, top curves). Login to comment
227 PG-01 produced large currents in both G551Dand G1349D-CFTR-expressing cells as shown in Fig. 6, A and B (bottom curves) and summarized in Fig. 6, C and D. Login to comment
229 In contrast, PG-01, SF-01, and the benzothiophene ⌬F508act-02 did not increase Cl- currents in G551Dand G1349D-CFTR-expressing cells (data not shown). Login to comment
283 The phenyglycines corrected defective gating in a number of CF-causing CFTR mutants including ⌬F508, G551D, G1349D, and D1152H. Login to comment
284 G551D and G1349D affect critical glycine residues in nucleotide binding domains 1 and 2 of CFTR, respectively (Hyde et al., 1990), producing a severe gating defect (Gregory et al., 1991; Logan et al., 1994; Derand et al., 2002; Zegarra-Moran et al., 2002). Login to comment
287 The potency for activation G1349D-CFTR by PG-01 was even better at ϳ40 nM. Login to comment

[hide] Differential sensitivity of the cystic fibrosis (C... J Biol Chem. 2006 Jan 27;281(4):1970-7. Epub 2005 Nov 25. Cai Z, Taddei A, Sheppard DN
Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2006 Jan 27;281(4):1970-7. Epub 2005 Nov 25., 2006-01-27 [PMID:16311240]

Abstract [show]
The genetic disease cystic fibrosis (CF) is caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Both mutants severely disrupt CFTR channel gating by decreasing mean burst duration (MBD) and prolonging greatly the interburst interval (IBI). To identify small molecules that rescue the gating defects of G551D- and G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551D- and G1349D-CFTR of phloxine B, pyrophosphate (PP(i)), and 2'-deoxy ATP (2'-dATP), three agents that strongly enhance CFTR channel gating. Phloxine B (5 microm) potentiated robustly G551D-CFTR Cl- channels by altering both MBD and IBI. In contrast, phloxine B (5 microm) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. PP(i) (5 mm) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl- channel. However, by altering both MBD and IBI, albeit with different efficacies, 2'-dATP (1 mm) potentiated both G551D- and G1349D-CFTR Cl- channels. Using the ATP-driven nucleotide-binding domain dimerization model of CFTR channel gating, we suggest that phloxine B, PP(i) and 2'-dATP alter channel gating by distinct mechanisms. We conclude that G551D- and G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific.

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No. Sentence Comment
1 Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Login to comment
3 To identify small molecules that rescue the gating defects of G551Dand G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551Dand G1349D-CFTR of phloxine B, pyrophosphate (PPi), and 2؅-deoxy ATP (2؅-dATP), three agents that strongly enhance CFTR channel gating. Login to comment
5 In contrast, phloxine B (5 ␮M) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. Login to comment
6 PPi (5 mM) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl-channel. Login to comment
7 However, by altering both MBD and IBI, albeit with different efficacies, 2؅-dATP (1 mM) potentiated both G551Dand G1349D-CFTR Cl-channels. Login to comment
9 We conclude that G551Dand G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific. Login to comment
26 To explore the mutation specificity of CFTR potentiators and to understand better their mechanism of action, the aim of the present study was to investigate the effects of the CFTR potentiators phloxine B, pyrophosphate (PPi), and 2Ј-deoxy-ATP (2Ј- dATP) on the CF mutants G551D and G1349D. Login to comment
28 Conversely, we selected the CF mutants G551D and G1349D because these mutants profoundly disrupt channel gating by affecting equivalent residues in the LSGGQ motifs of the two ATP-binding sites of CFTR (G551D, site 2, and G1349D, site 1) (4-6; 15-19) and because G551D is a common CF mutation (2). Login to comment
29 To quantify the efficacy with which the different CFTR potentiators restore normal channel gating to G551Dand G1349D-CFTR, we employed high resolution single-channel recording and * This work was supported by the Cystic Fibrosis Trust. Login to comment
39 MATERIALS AND METHODS Cells and Cell Culture-For this study, we used mouse mammary epithelial cells (C127 cells) stably expressing either wild-type human CFTR or the CF mutant G1349D and Fischer rat thyroid (FRT) epithelial cells stably expressing the CF mutant G551D.3 C127 cells were generous gifts of Professor M. J. Welsh (University of Iowa, Iowa City, IA) and Dr C. R. O`Riordan (Genzyme, Framingham, MA), whereas FRT cells were a generous gift of Drs. L. J. V. Galietta and O. Zegarra-Moran (Istituto Giannina Gaslini, Genoa, Italy). Login to comment
49 To determine the relationship between phloxine B concentration and channel activity for G551Dand G1349D-CFTR, we used membrane patches containing multiple active channels. Login to comment
50 For all other studies, we used membrane patches containing small numbers of active channels (wild-type and G1349D-CFTR, Յ4; G551D-CFTR, Յ6). Login to comment
54 Second, we used experimental conditions that robustly potentiate channel activity to determine the number of active channels in a membrane patch. For wild-type and G1349D-CFTR Cl-channels, channel numbers were counted in the presence of ATP (1 mM) and PKA (75 nM) alone. Login to comment
56 Despite our precautions, we cannot exclude the possibility of unobserved G551Dand G1349D-CFTR Cl-channels in membrane patches. Login to comment
57 Therefore, values of Po for G551Dand G1349D-CFTR might possibly be overestimated. Login to comment
67 Po was calculated from either open- and closed-times, as described previously (26), or by using the equation: Po ϭ I/͑n ϫ i͒ (Eq. 2) where n represents the number of active channels in the membrane patch. For wild-type CFTR, only membrane patches that contained a single active channel were used for burst analysis, whereas for G551Dand G1349D-CFTRs, we used membrane patches containing no more than four active channels. Login to comment
70 For example, for G1349D-CFTR, when n ϭ 1, MBD ϭ 34.4 Ϯ 7. Login to comment
73 4 Z. Cai, J.-H. Chen, and D. N. Sheppard, data not shown Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1971 and IBI í 2,090 Ϯ 481 ms (n ϭ 12) (p Ͼ 0.1 for both sets of data). Login to comment
88 RESULTS The Single-channel Activity of Wild-type, G551D-, and G1349D-CFTRs-Before investigating the rescue of the CF mutants G551D and G1349D by CFTR potentiators, we quantified their single-channel activity. Login to comment
89 Fig. 1A shows representative single-channel recordings of wild-type, G551D-, and G1349D-CFTR. Login to comment
90 Like other NBD mutants (3), G551Dand G1349D-CFTR were without effect on i but perturbed severely channel gating (Fig. 1, A and B). Login to comment
92 In contrast, G551Dand G1349D-CFTR both attenuated the duration of bursts and prolonged dramatically the interburst interval (Fig. 1A). Login to comment
93 As a result, the Po of G551Dand G1349D-CFTR were reduced markedly when compared with that of wild-type CFTR (Fig. 1C). Login to comment
95 Based on analyses of closed-time histograms (wild-type CFTR, ␶c ϭ 14.87 Ϯ 0.51 ms (n ϭ 10); G1349D-CFTR, ␶c ϭ 17.41 Ϯ 1.49 ms (n ϭ 5); p Ͼ 0.05), we used a burst delimiter (␶c) of 15 ms to discriminate inter-and intraburst closures.6 The MBD of G551Dand G1349D-CFTR were similar and both about one-third that of wild-type CFTR (Fig. 1D). Login to comment
96 In contrast, the IBI of G551Dand G1349D-CFTR were prolonged strikingly when compared with that of wild-type CFTR (Fig. 1E). Login to comment
97 Phloxine B Rescues the Gating Defect of G551D-CFTR but Not That of G1349D-CFTR-The fluorescein derivative phloxine B potentiates efficaciously wild-type and ⌬F508-CFTR Cl- currents (11). Login to comment
106 The single-channel activity of wild-type (WT), G551Dand G1349D-CFTRs. Login to comment
107 A, representative single-channel recordings of wild-type, G551D-, and G1349D-CFTRs in excisedinside-outmembranepatchesfromC127cellsexpressingwild-typeandG1349D-CFTRandFRTcellsexpressingG551D-CFTR.Inthisandsubsequentfigures,unlessotherwise indicated, ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution, voltage was -50 mV, and there was a large Cl-concentration gradient across the membrane patch (internal [Cl- ] ϭ 147 mM; external [Cl- ] ϭ 10 mM). Login to comment
109 For wild-type and G1349D-CFTRs, the membrane patches contained one and two active channels, respectively. Login to comment
111 B-E, i, Po, MBD, and IBI of wild-type, G551D-, and G1349D-CFTRs. Login to comment
112 Columns and error bars indicate means ϩ S.E. (wild-type, n ϭ 20 for Po and i, n ϭ 10 for MBD and IBI; G551D, n ϭ 35 for i, n ϭ 10 for Po, MBD, and IBI; G1349D, n ϭ 25 for i, Po, and MBD but n ϭ 5 for IBI). Login to comment
115 Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1972 tiates G551D-CFTR Cl- currents, we measured i, Po, MBD, and IBI in the absence and presence of the drug. Login to comment
121 Phloxine B (1-20 ␮M) also had biphasic effects on G1349D-CFTR Cl- currents with phloxine B (5 ␮M) potentiating the maximum current (Fig. 3, AandB)(11).Ofnote,themagnitudeofG1349D-CFTRCl- currentpoten- tiated by phloxine B (5 ␮M) was much smaller than that of G551D-CFTR but equivalent to that of wild-type CFTR (Figs. 2B and 3B). Login to comment
122 Because the single-channel activity of G1349D-CFTR is greatly diminished when com- paredwiththatofwild-typeCFTR(Fig.1),theseresultsindicatethatphlox- ine B fails to rescue the gating defect of G1349D-CFTR. Login to comment
123 Phloxine B caused a concentration-dependent reduction in i (Fig. 3C) but no change in the number of active G1349D-CFTR Cl-channels (n ϭ 8, Fig. 3A). Login to comment
124 Interestingly, G1349D-CFTR Cl-channels tended to open more frequently in the presence of phloxine B (Fig. 3A). Login to comment
126 However, the IBI of G1349D-CFTR in the presence FIGURE 2. Login to comment
138 Phloxine B potentiates weakly the single-channel activity of G1349D-CFTR. Login to comment
139 A, representative recordings show the effects of phloxine B (1 and 5 ␮M) on the activity of G1349D-CFTR Cl-channels. Login to comment
140 B, effects of phloxine B concentration on G1349D-CFTR Cl- currents (filled squares and solid line). Login to comment
143 C, effect of phloxine B concentration on i of G1349D- (filled squares and solid line) and wild-type CFTR (dotted line). Login to comment
145 D-F, effects of phloxine B (5 ␮M) on Po, MBD, and IBI of G1349D-CFTR Cl-channels. Columns and error bars indicate means ϩ S.E. (n ϭ 7). Login to comment
148 Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER JOURNAL OF BIOLOGICAL CHEMISTRY 1973 of phloxine B (5 ␮M) was over 5-fold longer than that of wild-type CFTR in the absence of drug. Login to comment
149 Thus, our data demonstrate that phloxine B rescues the gating defect of G551D-CFTR but not that of G1349D-CFTR. Login to comment
150 Effects of PPi on the Single-channel Activity of G551Dand G1349D-CFTR-The non-hydrolyzable inorganic phosphate analogue, PPi, enhances robustly wild-type CFTR Cl- currents by (i) increasing the rate of channel opening and (ii) decreasing markedly the rate of channel closure (12, 13). Login to comment
155 Like phloxine B (5 ␮M), PPi (5 mM) failed to potentiate the single-channel activity of G1349D-CFTR (Fig. 5). Login to comment
158 2Ј-Deoxy-ATP Activates Both G551Dand G1349D-CFTR Cl-Channels-Because both phloxine B and PPi failed to potentiate G1349D-CFTR, we searched for other agents that might rescue this CF mutant. Login to comment
180 Finally, we tested the effects of 2Ј-dATP on the single-channel activity of G1349D-CFTR.ReplacementofATP(1mM)by2Ј-dATP(1mM)waswith- 7 Comparable results were observed using the C1 7 O 7 C2 kinetic scheme (Ref. 28 and data not shown). Login to comment
187 Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1974 out effect on i but augmented G1349D-CFTR channel gating, leading to an increase in the number of active channels (Fig. 8). Login to comment
188 In the presence of 2Ј-dATP (1 mM), the Po and MBD of G1349D-CFTR increased 3.6- and 1.2-fold, respectively (Fig. 8, C and D), while IBI decreased by 54% (Fig. 8E). Login to comment
189 However, the MBD and IBI of G1349D-CFTR in the presence of 2Ј-dATP (1 mM) were 63% shorter and 2.6-fold longer, respectively, than those of wild-typeCFTRinthepresenceofATP(1mM;Figs.1,DandE,and8,Dand E). Login to comment
190 We interpret our data to suggest that 2Ј-dATP potentiates wild-type, G551D-, and G1349D-CFTR channel gating by similar mechanisms but that 2Ј-dATP incompletely restores normal channel gating to either G551D-CFTR or G1349D-CFTR. Login to comment
191 DISCUSSION The CF mutants G551D and G1349D affect equivalent residues in the LSGGQ motifs of NBD1 and NBD2. Login to comment
193 The CFTR potentiators phloxine B and 2Ј-dATP, but not PPi, augment G551D-CFTR channel gating, whereas only 2Ј-dATP enhances that of G1349D-CFTR. Login to comment
194 Our results demonstrate that G551Dand G1349D-CFTR have distinct pharmacological profiles. Login to comment
195 Molecular Mechanisms of CFTR Dysfunction in CF Previous studies demonstrated that G551Dand G1349D-CFTR cause a loss of Cl-channel function by disrupting ATP binding, hydrolysis, and thus, channel gating (17, 19, 31). Login to comment
196 Building on these data, our quantitative analysis of channel gating reveals that these mutants have exceptionally slow opening rates and very fast closing rates when compared with those of wild-typeCFTR.Toexplaintheseverityofthesegatingdefects,weconsider how G551Dand G1349D-CFTR might perturb the ATP-driven NBD dimerization model of CFTR channel gating (5, 20). Login to comment
197 The exceptionally slow rates of G551Dand G1349D-CFTR channel opening suggest that these mutants impede ATP binding to sites 1 and 2 with the result that the rate of NBD dimerization is retarded. Login to comment
198 Moreover, once the NBD dimer forms in G551Dand G1349D-CFTR Cl-channels, it is inherently unstable. Login to comment
212 Pyrophosphate (5 mM) fails to potentiate the single-channel activity of G1349D-CFTR. Login to comment
213 A, representative recordings show the effects of PPi (5 mM) on the activity of G1349D-CFTR Cl-channels. B-E, effects of PPi (5 mM) on i, Po, MBD, and IBI of G1349D-CFTR. Login to comment
215 Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1975 reduced. Login to comment
216 Of note, using a molecular model of the NBD dimer, Moran et al. (15) demonstrated that G551Dand G1349D-CFTR each destabilize ATP binding to sites 1 and 2. Login to comment
220 Below, we use the model of Vergani et al. (5) to discuss the effects of CFTR potentiators on wild-type, G551D-, and G1349D-CFTR. Login to comment
224 However, phloxine B also decreased the IBI of G551Dand G1349D-CFTR (present study), suggesting that the drug can promote NBD dimer formation for some, but not other, CF mutants (e.g. ⌬F508 (11)). Login to comment
226 G1349D-CFTR abolished the phloxine B-induced prolongation of channel openings, suggesting that G1349 either directly or indirectly contributes to the binding site for phlox- ineB.However,becausephloxineBdidnotpotentiatetheCFTRCl- chan- nel in the absence of ATP (11), we consider it unlikely that phloxine B interacts directly with site 1. Login to comment
236 G1349D-CFTR Cl-channels are potentiated by 2؅-dATP. Login to comment
237 A, single-channel recordings of G1349D-CFTR in the presence of either ATP (1 mM) or 2Ј-dATP (1 mM). Login to comment
239 B-E, effects of 2Ј-dATP on i, Po, MBD, and IBI of G1349D-CFTR, respectively. Login to comment
242 Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1976 the stability of the NBD dimer. Login to comment
247 Moreover, G1349D-CFTR abolished the potentiation of CFTR Cl- currents by PPi, suggesting that PPi might also bind to site 1 and accelerate channel opening by providing binding energy to drive NBD dimerization. Login to comment
248 However, because PPi cannot substitute for ATP in supporting CFTR channel gating (13) and because G1349D-CFTR has global effects on NBD dimer function (15), we suggest that the interaction of PPi with site 2 might energize NBD dimerization. Login to comment
254 First, among the agents that we tested, only 2Ј-dATP potentiated the gating behavior of both G551Dand G1349D-CFTR. Login to comment
255 We speculate that the reason why 2Ј-dATP rescued G1349D-CFTR channel gating, whereas phloxine B and PPi did not, is that 2Ј-dATP binds tightly to both sites 1 and 2. Login to comment

[hide] On the discovery and development of CFTR chloride ... Curr Pharm Des. 2006;12(4):471-84. Becq F
On the discovery and development of CFTR chloride channel activators.
Curr Pharm Des. 2006;12(4):471-84., [PMID:16472140]

Abstract [show]
Chloride channels play important roles in vital cellular signalling processes contributing to homeostasis in both excitable and non-excitable cells. Since 1987, more than ten ion channel genes have been identified as causing human hereditary diseases among them the genes for the voltage-dependent chloride channel ClC-1 (myotonia) and the cystic fibrosis transmembrane conductance regulator (CFTR) protein (cystic fibrosis). The CFTR gene was cloned in 1989 and its protein product identified as an ATP-gated and phosphorylation-regulated chloride channel during the following two years. Since then, searching for potent and specific small molecules able to modulate normal and mutated CFTR has become a crucial endpoint in the field for both our understanding of the physiological role that CFTR plays in epithelial cells and more importantly for the development of therapeutic agents to cure cystic fibrosis (CF). It is predicted that a pharmacological approach would help not only to restore the defective transport activity of mutant CFTR but also to correct the regulatory function of CFTR. This review describes the evolution of CFTR pharmacology and how during the last five years, high throughput screening assays have been developed to identify novel molecules, some of them probably constituting a reservoir of future therapeutic agents for CF.

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45 The glycine-to- aspartic acid missense mutations G551D and G1349D are class III mutations located within the signature sequence LSGGQ in NBD1 and LSHGH in NBD2, respectively [20]. Login to comment
207 By contrast, the response of the two mutants G1349D- and G551D-CFTR to genistein is dramatically altered [39]. Login to comment
208 Genistein is not able to stimulate G1349D- and CFTR bearing the double mutation G551D/G1349D whereas genistein stimulates G551D-CFTR [38, 39, 69] without any inhibition at high concentration [38, 39]. Login to comment

[hide] The glycine residues G551 and G1349 within the ATP... J Membr Biol. 2005 Dec;208(3):203-12. Epub 2006 Apr 7. Melin P, Norez C, Callebaut I, Becq F
The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B.
J Membr Biol. 2005 Dec;208(3):203-12. Epub 2006 Apr 7., [PMID:16604470]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.

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2 Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. Login to comment
4 However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 ± 1.13 lM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 ± 1.01 lM) but not activated by phloxine B. Finally, the double mutant G551D/ G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Login to comment
7 Key words: Cystic fibrosis - CFTR - ABC signature - G551D - G1349D - Phloxine B - Genistein - Non-Michaelis-Menten Introduction The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel belongs to the ATP-binding cassette (ABC) transporters, a large evolutionarily conserved family of integral membrane proteins that catalyze the active transport of a variety of solutes across biological membranes coupled to hydrolysis of nucleotides as a source of energy (Riordan et al., 1989; Vankeerberghen, Cuppens & Cassima, 2002). Login to comment
22 Individual delF508, G551D and G1349D mutations were created by site-directed mutagenesis as previously described (Melin et al., 2004). Login to comment
23 Double mutant G551D/G1349D (named 2GD-CFTR) was derived from pEGFP-G551D-CFTR. Login to comment
61 Results PROPERTIES OF G1349D-CFTR CHLORIDE CURRENT Individual delF508, G551D and G1349D and double G551D/G1349D (named 2GD-CFTR) mutations were created by site-directed mutagenesis and inserted into the pEGFP-C1-CFTR mammalian expression vector, allowing fusion of GFP to the N terminus of CFTR as previously described (Melin et al., 2004). Login to comment
64 The basal whole-cell current (mean current density of 14 ± 4 pA/pF at +40 mV, n = 3) was not significantly affected by the expression of G1349D-CFTR (mean current density of 12 ± 3 pA/pF at +40 mV, n = 7; Fig. 2A and D). Login to comment
65 The class III mutation G1349D, like G551D, impaired activation of CFTR channels by the adenylate cyclase activator forskolin (Fsk). Login to comment
66 The corresponding mean current densities are 11 ± 4 pA/pF for G551D-CFTR and 9 ± 4 pA/pF for G1349D-CFTR at +40 mV in the presence of 10 lM Fsk (n = 3). Login to comment
67 Using iodide efflux experiments, we tested increasing concentrations of Fsk from 0.1 to 100 lM but failed to stimulate any iodide efflux in G1349D-CFTR-expressing cells (n = 4 for each concentration, not shown). Login to comment
70 The cyclic adenosine monophosphate (cAMP)-activated G1349D-CFTR Cl) current was completely inhibited by 100 lM glibenclamide (Fig. 2C and D) (Sheppard & Welsh, 1993). Login to comment
71 Using iodide efflux experiments, we determined the concentration dependence of the effect of glibenclamide on G1349D-CFTR and G551D/ G1349D (noted 2GD) in cells activated by 10 lM Fsk + 100 lM IBMX. Login to comment
72 We calculated IC50 values of 8.3 ± 1.06 lM for G1349D-CFTR (n = 4; black symbols, Fig. 2E) and 5.5 ± 1.13 lM for 2GD-CFTR (n = 4; open symbols, Fig. 2E). Login to comment
75 We explored the effect of PhlxB on the Cl) channel activity of the following CFTR mutants: delF508, G551D, G1349D and 2GD. Login to comment
87 EFFECTS OF PHLXB ON G551D, G1349D AND G551D/G1349D CHANNELS In the next series of experiments, we examined the consequence of the glycine-to-aspartic acid mutations in NBD1 and NBD2 on the response to PhlxB in the presence of Fsk. Login to comment
91 Then, we examined the response to PhlxB of G1349D channels also in the presence of Fsk. Login to comment
98 found no evidence for stimulation of G1349D channels by increasing concentrations of PhlxB despite the presence of Fsk (Fig. 3B, black squares). Login to comment
99 The double mutant, carrying G551D and G1349D mutations, is also refractory to PhlxB stimulation (Fig. 3B, open squares). Login to comment
101 EFFECT OF PHLXB WHEN G1349D-, G551DAND 2GD-CFTR-EXPRESSING CELLS ARE STIMULATED BY FSK/IBMX Because the mutant G1349D can be activated by Fsk/IBMX but not by Fsk/PhlxB, we took advantage of this difference to investigate whether PhlxB could nevertheless inhibit the mutant channel after its activation by Fsk/IBMX, like the wt-CFTR channels. Login to comment
102 In the following experiments, we performed whole-cell patch-clamp experiments on COS-7 cells expressing G1349D-CFTR stimulated by Fsk/ IBMX and subsequently challenged by application of 10 lM PhlxB into the bath. Login to comment
106 After stable activation of the G1349D-CFTR current, addition of 10 lM PhlxB (in the continuous presence of Fsk/IBMX) inhibited G1349D-CFTR currents (Fig. 4A and B). Login to comment
108 Further application of 100 lM glibenclamide in the extracellular bath had no additional inhibitory effect (Fig. 4A), confirming that the G1349D-CFTR current was totally inhibited by PhlxB. Login to comment
112 However, contrary to G1349D-CFTR, PhlxB did not inhibit G551D-CFTR currents after stable activation of G551D (compare left and right whole-cell 0 100 200 300 400 -1000 0 1000 2000 3000 I(pA) Time (ms) 0 100 200 300 400 -1000 0 1000 2000 3000 I(pA) Time (ms) 0 100 200 300 400 -1000 0 1000 2000 3000I(pA) Time (ms) Fsk + IBMXbasal Fsk + IBMX + Glib -100-80 -60 -40 -20 20 40 60 80 100 -1000 1000 2000 basal Fsk+IBMX +Glib Vm (mV) I (pA) -6 -5 -4 0 25 50 75 100 2GD G1349D Log [glibenclamide] (M) %ofactivation ns ns ns ns ns A B D E C Fig. 2. Login to comment
113 Activation by cAMP cocktail and inhibition by glibenclamide of G1349D-CFTR channels. Login to comment
117 Vm, membrane voltage; (E) Concentration- responses curves of glibenclamide on COS-7 cells transfected with GFP-G1349D-CFTR and 2GD after maximal activation with cAMP cocktail. Login to comment
120 IC50 values calculated in these conditions are for G1349D channels, 8.3 ± 1.06 lM, and for 2GD channels, 5.55 ± 1.13 lM. Login to comment
126 We also noted two differences between G551D and G1349D-CFTR channels stimulated by Fsk+IBMX. Login to comment
127 First, the time to maximal current stimulation with G551D-CFTR, 120 ± 9 s (n = 4), was significantly faster (P < 0.01) than that for G1349D (194 ± 8 s, n = 4). Login to comment
128 Second, the current density measured at +40 mV was 26 ± 5 pA/pF (n = 6) for G551D construct, a value approximately threefold smaller (P < 0.001) than the current density measured for G1349D, 88 ± 13 pA/pF (n = 4). Login to comment
129 Finally, we determined the concentration-dependent inhibitory effects of PhlxB and genistein on G1349D and 2GD-CFTR channels after stimulation by Fsk/IBMX (Fig. 6). Login to comment
131 We calculated the Ki for PhlxB on G1349D (40 ± 1.01 lM, n = 4 for each concentration; Fig. 6A, black symbols). Login to comment
133 With genistein, the calculated Ki was 121 ± 1.14 lM for G1349D channels (n = 4 for each concentration; Fig. 6B, black symbols). Login to comment
142 In this configuration, each nucleotide is sandwiched between the two NBDs and each nucleotide-binding site is formed by the Walker A, Walker B, Q-loop and wt-CFTR delF508 (24h/27˚C) -7 -6 -5 -4 0.00 0.05 0.10 0.15 0.20 Log [PhlxB] (M) kpeak-kbasal(min-1 ) ** ns ns ** * -7 -6 -5 -4 0.00 0.05 0.10 0.15 0.20 G551D G1349D 2GD Log [PhlxB] (M) kpeak-kbasal(min-1 ) A B Fig. 3. Login to comment
147 (B) Concentration-response curves of PhlxB on COS-7 cells transiently transfected with GFP-CFTR with the following mutations: G551D, -G1349D and -2GD. Login to comment
151 Kinetic parameters for activation (Ks) and inhibition (Ki) of CFTR by PhlxB and genisteina PhlxB Genistein Construct Ks Ki Ks Ki wt-CFTR 3.2 ± 1.6 38 ± 1.4 10 ± 1b 106 ± 1b delF508 3 ± 1.8 33 ± 1 9.8 ± 1b 107 ± 1b G551D 2 ± 1.13 -d 13 ± 1.25b - G1349D - 40 ± 1.01c - 121 ± 1.14c 2GD - - - - a All data (in lM) are n = 4. b Data from Melin et al., 2004. c With activation by Fsk/IBMX. Login to comment
158 Interestingly our data show that G1349D-CFTR elicited a larger Cl) current than G551D-CFTR, these two glycine residues being located within the NBD1 and NBD2 active sites, respectively (Fig. 1B). Login to comment
161 Effects of PhlxB on cAMP-stimulated G1349D-CFTR channels. Login to comment
162 (A) Representative time course of G1349D-CFTR current amplitude in the presence of various agonists. Login to comment
163 (B) Representative trace of current recorded on G1349D channels with Fsk + IBMX (*) and with Fsk + IBMX + PhlxB (**). Login to comment
164 (C) I/V relationships for G1349D-CFTR chloride current (mean ± SEM) normalized by cell capacitance. Login to comment
167 (D) Summary of current amplitudes (mean ± SEM between +40 and )40 mV) recorded on cells transfected with GFP-CFTR- G1349D in various conditions: basal (n = 4), Fsk + IBMX (n = 4), Fsk + IBMX + PhlxB (n = 4), Fsk + IBMX + PhlxB + glibenclamide (Glib, n = 4). Login to comment
171 According to these data, we show that the LSGDQ mutated sequence (with G551D) drastically impairs activation of CFTR Cl) channel activity by Fsk/IBMX compared to the LSHDH mutated sequence (with G1349D). Login to comment
174 Drugs like genistein and PhlxB are associated with direct binding at the NBDs (Randak et al., 1999; Cai & Sheppard, 2002), consistent with our observations that CFTR modulation is altered by NBD mutations G551D and G1349D. Login to comment

[hide] CFTR chloride channel drug discovery--inhibitors a... Curr Pharm Des. 2006;12(18):2235-47. Verkman AS, Lukacs GL, Galietta LJ
CFTR chloride channel drug discovery--inhibitors as antidiarrheals and activators for therapy of cystic fibrosis.
Curr Pharm Des. 2006;12(18):2235-47., [PMID:16787252]

Abstract [show]
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cAMP-activated chloride channel expressed in epithelia in the lung, intestine, pancreas, testis and other tissues, where it facilitates transepithelial fluid transport. In the intestine CFTR provides the major route for chloride secretion in certain diarrheas. Mutations in CFTR cause the hereditary disease cystic fibrosis, where chronic lung infection and deterioration in lung function cause early death. CFTR is a well-validated targeted for development of inhibitors for therapy of secretory diarrheas and activators for therapy in cystic fibrosis. Our lab has identified and optimized small molecule inhibitors of CFTR, as well as activators of DeltaF508-CFTR, the most common mutant CFTR causing cystic fibrosis. High-throughput screening of small molecule collections utilizing a cell-based fluorescence assay of halide transport yielded thiazolidinone and glycine hydrazide CFTR inhibitors that block enterotoxin-mediated secretory diarrhea in rodent models, including a class of non-absorbable inhibitors that target the CFTR pore at its external entrance. Benzothiophene, phenylglycine and sulfonamide potentiators were identified that correct the defective gating of DeltaF508-CFTR chloride channels, and other small molecules that correct its defective cellular processing. Small molecule modulators of CFTR function may be useful in the treatment of cystic fibrosis, secretory diarrhea and polycystic kidney disease.

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221 Phenylglycines had another interesting property in being able to activate other CFTR mutants including G551D, G1349D, and D1152H (Fig. 7C). Login to comment
242 C. Apical membrane current in FRT cells expressing G1349D-CFTR showing responses to forskolin and genistein or PG-01. Login to comment

[hide] Functional analysis of mutations in the putative b... J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23. Zegarra-Moran O, Monteverde M, Galietta LJ, Moran O
Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition.
J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23., 2007-03-23 [PMID:17244607]

Abstract [show]
An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg(553), Ala(554), and Val(1293). The activity of CFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.

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37 Printed in the U.S.A. 9098 tion that mutations in conserved residues of the NBDs such as G551D and G1349D exhibit a shift in the affinity for potentiators (5, 15-18). Login to comment
41 After in silico docking of several compounds, we compared the theoretical binding free energy measured on the model, with the experimental binding free energy obtained from dissociation constants from wild type, G551D, and G1349D proteins. We found a good correlation between these two parameters for a putative binding site located in the interface of the NBD1-NBD2 dimer, embedded in a cavity on NBD1, and interacting also with the NBD2 surface. Login to comment
186 Probably, the NBD with higher affinity for potentiators is NBD1, because CF mutation G551D, situated on NBD1 near the potentiator binding site, causes a more pronounced effect on the equilibrium constant for the activation site than the symmetrical CF mutation on NBD2, G1349D (16). Login to comment

[hide] G551D and G1349D, two CF-associated mutations in t... J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12. Bompadre SG, Sohma Y, Li M, Hwang TC
G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects.
J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12., [PMID:17353351]

Abstract [show]
Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po approximately 10-fold lower than WT. Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.

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1 (c) The Rockefeller University Press $15.00 Volume 129 Number 4 April 2007 285-298 http://www.jgp.org/cgi/doi/10.1085/jgp.200609667 285 A RT I C L E G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects Silvia G. Bompadre,1,2 Yoshiro Sohma,2,3 Min Li,1,2 and Tzyh-Chang Hwang1,2 1Department of Medical Pharmacology and Physiology, 2Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 3Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). Login to comment
5 On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Login to comment
12 G1349D-CFTR maintains ATP dependence, albeit with a Po ‫-01ف‬fold lower than WT. Login to comment
13 Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. Login to comment
14 G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Login to comment
25 These mutations can be divided into four classes based on the mechanisms that disrupt CFTR function (Welsh and Smith, 1993): defective protein production (I); defective protein processing (II); defective activation and regulation (III), including G551D and G1349D; and defective conductance (IV). Login to comment
32 However, the G1349D mutation is associated with a milder clinical phenotype (Brancolini et al., 1995, Salvatore et al., 2002). Login to comment
33 Although G551D-CFTR and G1349D-CFTR have been used in pharmacological studies for years (e.g., Illek et al., 1999; Galietta et al., 2001; Moran et al., 2005; Pedemonte et al., 2005), little is known about the mechanism responsible for their dysfunction due to limited functional studies of these two mutants at a single-channel level (e.g., Cai et al., 2006). Login to comment
37 Logan et al. (1994) reported that as a consequence of the G551D, or G1349D mutation, nucleotide binding to isolated recombinant NBD1 and NBD2 is decreased. Login to comment
39 Wilkinson et al. (1996) measured the rates of activation and deactivation of macroscopic CFTR currents in Xenopus oocytes and found that the apparent on rate of channel activation by cAMP for G551D-CFTR or G1349D-CFTR was drastically reduced. Login to comment
42 We have studied the response of G551D-CFTR and G1349D-CFTR to ATP, ADP, and AMP-PNP in excised inside-out membrane patches. Login to comment
44 The G551D mutation completely abolishes the response of the channel to ATP and ADP, whereas the G1349D mutation remains responsive to ATP and the ATP-induced activity can be inhibited by ADP. Login to comment
46 Interestingly, compared with WT data, the ATP dose-response relationship for G1349D-CFTR is less steep and shows an increase of the apparent affinity for ATP. Login to comment
47 These features of the ATP dose response for G1349D-CFTR could be reproduced by assuming that ATP binding to ABP1, where the mutation is located, hinders channel opening. Login to comment
50 Point mutations (G551D and G1349D) were introduced into WT-CFTR by QuikChange XL method (Stratagene). Login to comment
60 Thus the G551D mutation is located in ABP2 and the G1349D mutation is located in ABP1. Login to comment
102 AMP-PNP was purchased from Roche and stored as 250 mM stock in H2O at -20°C. R E S U LT S Expression of G551D-CFTR and G1349D-CFTR It has been previously reported that neither G551D-CFTR nor G1349D-CFTR exhibit trafficking defects in COS cells (Gregory et al., 1991). Login to comment
105 G551D-CFTR and G1349D-CFTR expression in CHO cells. Login to comment
106 (A) Western blot analysis for WT-, G551D-, and G1349D-CFTR. Login to comment
110 (B) Mean current densities obtained from whole-cell experiments for G551D (n = 30), G1349D (n = 29), and WT (n = 20). Login to comment
111 Columns and error bars indicate means ± SEM, * indicates P < 0.01 between G551D and G1349D. Login to comment
119 Fig. 2 B shows that the current densities of G551D (n = 30) and G1349D (n = 29) mutants are lower than that of WT-CFTR (n = 20). Login to comment
120 However, the current density of G1349D-CFTR is significantly larger than that of G551D-CFTR (P < 0.01), suggesting that the Po of G1349D-CFTR is higher than that of G551D-CFTR. Login to comment
121 ATP-dependent Gating of G551D-CFTR and G1349D-CFTR To investigate the mechanism responsible for the different gating behavior of G551D and G1349D mutants, we studied both mutants in excised inside-out membrane patches. Login to comment
125 Gating of G551D-CFTR and G1349D-CFTR in excised inside-out membrane patches. Login to comment
130 (C) A recording of G1349D-CFTR channel currents in an excised inside-out membrane patch. Login to comment
135 (E) Comparisons of mean macroscopic current amplitude for G551D- (n = 16), G1349D- (n = 18), and WT-CFTR (n = 13). Login to comment
144 When the same experiment was performed with G1349D-CFTR, we observed generally larger currents than that of G551D-CFTR (e.g., Fig. 3 C). Login to comment
145 In addition, unlike G551D-CFTR, G1349D-CFTR channel current decreases immediately upon ATP washout, a property shared with the WT channels (Fig. 3 D). Login to comment
146 Note that for both WTand G1349D-CFTR a small but significant fraction of the current remained for minutes after nucleotide removal. Login to comment
148 The steady-state mean current amplitude (a product of the number of functional channels, Po and single-channel amplitude) shows a similar pattern as that of whole-cell recordings (i.e., WT > G1349D > G551D). Login to comment
153 Since the Po for WT channels in the presence of 1 mM ATP is 0.45 ± 0.04 (see Fig. 9 D), the estimated Po are ≅ 0.0037 for G551D (120 times smaller than the WT Po), and ≅ 0.045 (10 times smaller than the WT Po) for G1349D. Login to comment
155 Comparison of the mean open times of G551D-, G1349D-, and WT-CFTR. Login to comment
156 (A) Expanded single-channel current traces in the presence of 1 mM ATP for G551D-, G1349D-, and WT-CFTR. Login to comment
168 It is interesting to note that Cai et al. (2006) found that the G551D and G1349D mutations shorten the open time of the channels. Login to comment
170 Since free [Mg2+] is known to affect the open time of WT-CFTR, presumably by altering the ATP hydrolysis rate (Dousmanis et al. 2002), we next examined the effect of Mg2+ on the closing rate of G551Dand G1349D-CFTR channels. Login to comment
175 Surprisingly, we also did not observe an increase in the open time in G1349D channels, although some ATP dependence is retained for this mutant (n = 6; unpublished data). Login to comment
176 Effect of ADP and AMP-PNP on G551D-CFTR and G1349D-CFTR Channels The results shown above indicate that G551D and G1349D, mutations at the equivalent position in the signature sequence of NBD1 and NBD2, respectively, exhibit different gating defects. Login to comment
182 In contrast, G1349D-CFTR channels, like WT-CFTR, can be inhibited by ADP; interestingly however, the level of inhibition was slightly but significantly less (P < 0.001), ‫04ف‬ ± 3% (n = 8, Fig. 6 C). Login to comment
185 In excised patches, after activation with 1 mM ATP + PKA, we applied 2 mM AMP-PNP + 1 mM ATP to WT (Fig. 7 A), G551D (Fig. 7 B), Figure 6. Effect of ADP on G551D, G1349D, and WT-CFTR. Login to comment
186 Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Login to comment
187 Currents from both WTand G1349D-CFTR are reduced by ADP, but ADP fails to inhibit G551D-CFTR channel currents. Login to comment
188 (D) Summary of percent inhibition by 500 μM ADP for G551D- (n =10), G1349D- (n = 8), and WT-CFTR (n = 8). Login to comment
190 * indicates P < 0.001 between G1349D and WT. Login to comment
191 and G1349D channels (Fig. 7 C). Login to comment
196 In contrast, AMP-PNP failed to increase either the G551D- or G1349D-CFTR currents (Fig. 7, B and C). Login to comment
197 Upon removal of the nucleotides, most of the G1349D-CFTR channel currents decreased rapidly (Fig. 7 C). Login to comment
199 [ATP] Dependence of G551Dand G1349D-CFTR Our data suggest that the G551D mutation abolished nucleotide-dependent gating. Login to comment
206 We next examined the ATP dependence of G1349D-CFTR more thoroughly by measuring the activity of the channel at different [ATP]. Login to comment
207 In excised patches, G1349D-CFTR channels were exposed to 2.75 mM ATP + PKA, and then to various ATP concentrations (from 2 μM to 10 mM). Login to comment
208 The experiments were performed by quantifying G1349D-CFTR channel current at a test [ATP] that was bracketed with 2.75 mM ATP to ensure the absence of a time-dependent rundown (for details see Zeltwanger et al., 1999). Login to comment
209 Fig. 9 (A and B) shows representative traces for WTand G1349D-CFTR. Login to comment
210 Interestingly, the magnitude of current elicited by 50 μM ATP, relative to that with 2.75 mM ATP, is larger for G1349D-CFTR (Fig. 9 B) than for WT-CFTR (Fig. 9 A), suggesting that G1349D-CFTR channels are "more sensitive" to ATP than WT-CFTR. Login to comment
211 Fig. 9 C shows the normalized ATP dose response for G1349D-CFTR with an overlaid WT-CFTR data for comparison (from Zhou et al., 2006). Login to comment
212 Interestingly, similar to that of WT-CFTR, the G1349D-CFTR channel activity is saturated at millimolar [ATP], but the ATP dose-response relationship is shifted to the left. Login to comment
215 Therefore, the difference in the dose response between WTand G1349D-CFTR (apparent Kd and maximal Po) reflects effects of the mutation on the opening rate of the channel. Login to comment
216 More interestingly, the dose-response curve of G1349D-CFTR is not as steep as that of WT-CFTR. Login to comment
217 At low micromolar [ATP], the relative fraction of G1349D current is higher than that of WT. Login to comment
218 Qualitatively speaking, as the [ATP] is increased, the activity of the G1349D mutant does not increase as much as that of WT channels, as if the increasing activation of G1349D-CFTR upon Figure 7. Effect of AMP-PNP on G551D, G1349D, and WT-CFTR. Login to comment
219 Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Login to comment
221 Neither G551D- nor G1349D-CFTR responds to AMP-PNP (n = 4 each). Login to comment
223 Kinetic Modeling for G1349D-CFTR To explain the unusual ATP dose-response relationship of G1349D-CFTR, we resort to CFTR gating schemes that incorporate two ATP binding sites. Login to comment
231 We then tried to fit the G1349D data using this scheme. Login to comment
232 To fit the G1349D data we need to be able to reproduce the shift of the curve, the flattening of the slope, as well as an ‫-01ف‬fold reduced maximal Po. Login to comment
233 Since the open time for G1349D-CFTR is not significantly different from that of WT channels, kc(hydro) remains unchanged. Login to comment
244 ATP dose-response for G1349D-CFTR. Login to comment
245 Representative traces of WT- (A) and G1349D-CFTR (B) channels in response to 50 μM and 2.75 mM ATP. Login to comment
246 Note that the amount of current elicited by 50 μM ATP, relative to the amount of current elicited by 2.75 mM ATP, is larger for G1349D (ratio = 0.65) than for WT (ratio = 0.42) in these particular patches. Login to comment
247 (C) Normalized ATP dose-response relationship for G1349D-CFTR (red circles) and WT-CFTR (blue squares, from Zhou et al., 2006). Login to comment
248 (D) Relationships between [ATP] and single-channel Po (blue squares for WT, from Zhou et al., 2006, and red open circles for G1349D). Login to comment
249 The G1349D Po was calculated under the assumption that the maximal Po is 10 times lower than WT Po. Login to comment
259 We calculated the open probability of G1349D-CFTR for this expanded model using the same kinetic parameters listed in Table I with two added new parameters (kc(SPT) = 2 s-1, kO(SPT) = 0.006 s-1; fromt Bompadre et al., 2005b). Login to comment
260 Adding this ATP-independent component could not replicate the overall shape of the ATP dose response for G1349D-CFTR (unpublished data). Login to comment
261 We then tested the possibility that the G1349D mutation may affect the opening rate for the spontaneous openings. Login to comment
264 On the other hand, when kc(SPT) and kO(SPT) were kept the same as those of WT channels, the ratio of the G1349D-CFTR current produced by the ATP-independent openings to the maximal current (0.057 ± 0.010) is very close to what the model predicts (0.06). Login to comment
265 We should point out that even though we could not simulate the flattening of the dose-response relationship for G1349D-CFTR by modifying the opening rate for the spontaneous openings, Kd2, and the ATP-dependent opening rate (kO(2ATP)), we cannot completely rule out the possibility that some combinations of these parameters could produce a curve similar to our data. Login to comment
274 (C) Normalized ATP dose response for G1349D-CFTR channels (red circles) and different simulated results obtained with Scheme A. Login to comment
279 The parameter sets A, B, and C correspond to different attempts to fit the G1349D-CFTR data (green, brown, and red lines in Fig. 10 C, respectively). Login to comment
283 Using Scheme B, we can also obtain a dose-response relationship that reproduces all the features of the G1349D dose response in at least two different ways (Table II). Login to comment
284 First, without changing any of the WT binding parameters for ABP1 or ABP2, simply reducing kO (1ATP) to 1 s-1 and kO (2ATP) to 0.16 s-1 can produce data strikingly similar to those of G1349D-CFTR (green line in Fig. 11 C). Login to comment
285 The dose-response curve of G1349D-CFTR can also be successfully reproduced by slightly decreasing the affinity for ATP at ABP2 (e.g., by increasing Kd2 to 280 μM) and simultaneously lowering the opening rate kO (2ATP) to 0.16 s-1 (red line in Fig. 11 C). Login to comment
286 These results thus suggest that the G1349D mutation decreases the channel opening rate when ABP1 is occupied by ATP (i.e., kO(2ATP)). Login to comment
290 On the other hand, G1349D-CFTR exhibits a fairly different behavior than G551D-CFTR; it maintains some ATP dependence, but with a lower Po than WT channels due to a lower opening rate. Login to comment
303 (B) Dose-response relationships for WT (red squares) and G1349-CFTR (red circles) and calculated data based on Scheme B and the parameters summarized in Table II TA B L E I I A Summary of the Kinetic Parameters used in Fig. 11 for Scheme B Scheme A WT G1349D (a) G1349D (b) Kd1 (μM) 10 10 10 Kd2 (μM) 130 130 280 kO(1ATP) (s-1) 2.5 1 2.5 kO(2ATP) (s-1) 2.5 0.16 0.16 kC(hydro) (s-1) 3 3 3 kO(SPT) (s-1) 0.006 0.006 0.006 kC(SPT) (s-1) 2 2 2 These sets of parameters correspond to the fits obtained for WT-CFTR (blue line in Fig. 11 B) and G1349D-CFTR (a corresponds to the green line in Fig. 11 C; b corresponds to the red line in Fig. 11 C). Login to comment
332 In contrast to G551D-CFTR, G1349D-CFTR still maintains some ATP dependence, but has a maximal Po ‫-01ف‬fold lower than WT-CFTR. Login to comment
333 It is interesting to note that the inhibition of G1349D-CFTR channels by ADP is slightly smaller than the inhibition of WT channels. Login to comment
334 To explain this observation we have to realize that G1349D channels also have ATP-independent openings, and because of a much lower Po at maximal [ATP], the fraction of the total current that originates from the ATP-independent openings is relatively higher in G1349D than in WT channels. Login to comment
335 Since the ATP-independent activity is insensitive to ADP, the portion of the total current that can be inhibited by ADP is relatively smaller in G1349D than WT channels. Login to comment
336 If binding of ATP to ABP1 is not essential for channel opening as described above, why does the G1349D mutation, located at ABP1, decrease the maximal Po? Login to comment
337 Since the open time of the mutant channel does not differ significantly from that of WT channels, we can conclude that the defect of G1349D-CFTR resides in a lowered opening rate. Login to comment
339 The most intriguing feature of G1349D-CFTR is the flattened ATP dose-response curve, suggesting a negative cooperativity between the two ATP binding sites. Login to comment
340 Based on the results of our modeling, we propose that this negative cooperativity seen with the G1349D mutation arises from a negative effect on channel opening by ATP binding at ABP1. Login to comment
348 It is interesting to note that AMP-PNP does not lock open either G551D or G1349D channels. Login to comment
352 It is however puzzling that AMP-PNP is ineffective on G1349D-CFTR. Login to comment
353 Interestingly, Cai et al. (2006) reported that pyrophosphate, an agent that potentiates CFTR by a similar mechanism as AMP-PNP, also fails to act on G1349D-CFTR. Login to comment
354 Perhaps, the aspartate side chain of G1349D-CFTR prevents the formation of a stable locked-open conformation. Login to comment
355 A similar mechanism may also account for the lack of effect on the open time of G1349D-CFTR by removing free Mg2+. Login to comment
356 In addition to the mechanistic insights into how CFTR`s two NBDs work concertedly to gate the channel, our observations also provide a quantitative explanation for the different phenotypes exhibited in CF patients carrying the G551D or G1349D mutation. Login to comment
365 Nevertheless, it is interesting to note that 20 μM genistein increases G1349D-CFTR current density 6.8 ± 1.9-fold (n = 14) compared with forskolin alone. Login to comment
366 With this increase, G1349D current density is ‫%06ف‬ of that of WT, a level likely sufficient to completely restore the physiological function of chloride secretion. Login to comment
368 This may be adequate to reach a level of activity similar to that of G1349D-CFTR but unlikely to completely restore the physiological function of this mutant channel. Login to comment
413 2006. Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment

[hide] Structure-activity relationship of 1,4-dihydropyri... Mol Pharmacol. 2007 Jul;72(1):197-207. Epub 2007 Apr 23. Pedemonte N, Boido D, Moran O, Giampieri M, Mazzei M, Ravazzolo R, Galietta LJ
Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel.
Mol Pharmacol. 2007 Jul;72(1):197-207. Epub 2007 Apr 23., [PMID:17452495]

Abstract [show]
Mutations occurring in the CFTR gene, encoding for the cystic fibrosis transmembrane conductance regulator chloride channel, cause cystic fibrosis (CF). Mutations belonging to class II, such as DeltaPhe508, give rise to a protein with both a defective maturation and altered channel gating. Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. We have previously identified antihypertensive 1,4-dihydropyridines (DHPs), a class of drugs that block voltage-dependent Ca(2+) channels, as effective potentiators of CFTR gating, able to correct the defective activity of CFTR mutants (Mol Pharmacol 68:1736-1746, 2005). However, optimization of potency for CFTR versus Ca(2+) channels is required to design selective compounds for CFTR pharmacotherapy. In the present study, we have established DHP structure-activity relationship for both CFTR potentiation and Ca(2+) channel inhibition using cell-based assays for both types of channels. A panel of 333 felodipine analogs was studied to understand the effect of various substitutions and modifications in the DHP scaffold. Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca(2+) channels and strong effect as potentiators on the DeltaPhe508, G551D, and G1349D CFTR mutants.

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2 Mutations belonging to class III, such as G551D and G1349D, cause only a gating defect. Login to comment
7 Our results show that alkyl substitutions at the para position of the 4-phenyl ring lead to compounds with very low activity on Ca2ϩ channels and strong effect as potentiators on the ⌬Phe508, G551D, and G1349D CFTR mutants. Login to comment
28 Other class III mutations, such as G1349D, are much rarer. Login to comment
34 Conversely, phenylglycines, another class of compounds identified by high-throughput screening, are effective also on G551D and G1349D channels. Login to comment
45 To this purpose, we screened a set of 333 felodipine analogs using cell-based assays to determine their activity on three CFTR mutants (⌬Phe508, G551D, and G1349D) and on DHP-sensitive Ca2ϩ channels. Login to comment
48 Fischer rat thyroid (FRT) cells were stably transfected with ⌬Phe508, G551D, or G1349D-CFTR, and the halide-sensitive yellow fluorescent protein mutant YFP-H148Q/I152L (Galietta et al., 2001a). Login to comment
66 Measurements of CFTR activity were carried out on FRT cells expressing mutant CFTR and the halide-sensitive YFP 24 h (G551D and G1349D) or 48 h (⌬Phe508) after plating on microplates. Login to comment
124 The panel of compounds was tested on FRT cells coexpressing the halide-sensitive mutant H148Q/I152L of the fluorescent protein YFP and the CFTR mutants ⌬Phe508, G551D, and G1349D. Login to comment
130 A, chemical structure of tested compounds. B, representative fluorescence traces showing the response to I- addition in FRT cells expressing G1349D-CFTR under resting conditions (saline) or upon stimulation with forskolin alone (20 ␮M) or in the presence of test compounds. Login to comment
187 Conversely, the G1349D mutant displayed a sensitivity between that of ⌬Phe508 and G551D (Fig. 8, B and C). Login to comment
194 On the other hand, the same DHPs maintained a good activity on CFTR channels with an apparent Ka for the ⌬Phe508 and G1349D mutants in the range 100 to 700 nM (Fig. 9, C and E). Login to comment
206 In particular, CFTR potentiators are needed to restore activity in those CFTR mutants affected by impaired channel gating (class III mutants), like G551D and G1349D. Login to comment
214 Note that the concentrations needed to stimulate the G551D mutant are always 10 to 20 times higher than those effective on ⌬Phe508 or G1349D. Login to comment
223 In parallel, we have used the functional assay based on the halide-sensitive YFPs to measure activity of compounds on ⌬Phe508, G551D, and G1349D CFTR mutants. Login to comment
225 Our first result is that DHP-based structures activate also G1349D-CFTR, besides ⌬Phe508 and G551D mutants. Login to comment
228 ⌬Phe508 and G1349D were activated at nanomolar concentrations by the most potent compounds. Login to comment
237 B-E, dose-response relationships obtained for indicated compounds on VDCC (B), ⌬Phe508-CFTR (C), G551D-CFTR (D), and G1349D-CFTR (E). Login to comment

[hide] Discovery of alpha-aminoazaheterocycle-methylglyox... J Pharmacol Exp Ther. 2007 Sep;322(3):1023-35. Epub 2007 Jun 19. Routaboul C, Norez C, Melin P, Molina MC, Boucherle B, Bossard F, Noel S, Robert R, Gauthier C, Becq F, Decout JL
Discovery of alpha-aminoazaheterocycle-methylglyoxal adducts as a new class of high-affinity inhibitors of cystic fibrosis transmembrane conductance regulator chloride channels.
J Pharmacol Exp Ther. 2007 Sep;322(3):1023-35. Epub 2007 Jun 19., [PMID:17578899]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl(-) channel in the apical membrane of epithelial cells for cAMP-dependent Cl(-) secretion. Here we report on the synthesis and screening of a small library of nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl(-) channels. In whole-cell patch-clamp experiments of Chinese hamster ovary (CHO) cells expressing WT-CFTR, we recorded rapid and reversible inhibition of forskolin-activated CFTR currents in the presence of the adducts 5a and 8a,b at 10 pM concentrations. Using iodide efflux experiments, we compared concentration-dependent inhibition of CFTR with glibenclamide (IC(50) = 14.7 microM), 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-t hiazolidinone (CFTR(inh)-172) (IC(50) = 1.2 microM), and alpha-aminoazaheterocycle-methylglyoxal adducts and identified compounds 5a (IC(50) = 71 pM), 8a,b (IC(50) = 2.5 nM), and 7a,b (IC(50) = 3.4 nM) as the most potent inhibitors of WT-CFTR channels. Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). No effect of compound 5a was detected on the volume-regulated or calcium-regulated iodide efflux. Picomolar inhibition of WT-CFTR with adduct 5a was also found using a 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescent probe applied to the human tracheobronchial epithelial cell line 16HBE14o-. Finally, we found comparable inhibition by 5a or by CFTR(inh)-172 of forskolin-dependent short-circuit currents in mouse colon. To the best of our knowledge, these new nontoxic alpha-aminoazaheterocycle-methylglyoxal adducts represent the most potent compounds reported to inhibit CFTR chloride channels.

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1 Here we report on the synthesis and screening of a small library of nontoxic ␣-aminoazaheterocycle-methylglyoxal adducts, inhibitors of wild-type (WT) CFTR and G551D-, G1349D-, and F508del-CFTR Cl-channels. Login to comment
4 Similar ranges of inhibition were also found when these compounds were evaluated on CFTR channels with the cystic fibrosis mutations F508del (in temperature-corrected human airway epithelial F508del/ F508del CF15 cells)-, G551D-, and G1349D-CFTR (expressed in CHO and COS-7 cells). Login to comment
25 Here we present the synthesis and identification of ␣-aminoazaheterocycle-methylglyoxal adducts as highly potent inhibitors of wild-type CFTR and CFTR affected by the following CF mutations: F508del, G551D, and G1349D. Login to comment
75 The G1349D mutation was created by site-directed mutagenesis as described previously (Melin et al., 2004) and transiently expressed in COS-7 cells, 12 to 24 h after seeding, using cationic lipids (jetPEI; Qbiogene Inc., Carlsbad, CA) with 1 ␮g/ml of plasmid. Login to comment
116 CFTR-dependent iodide efflux was stimulated either by forskolin (WT-CFTR) or by a cocktail containing forskolin with genistein (F508del- and G551D-CFTR) or with the benzo[c]quinolizinium derivative MPB-91 (G1349D-CFTR). Login to comment
190 The present study was undertaken on the following cells: CHO cells overexpressing WT-CFTR or mutated G551D-CFTR and COS-7 cells expressing G1349D-CFTR (both G551D and G1349D are class III CF mutations); two human airway epithelial cells, Calu-3 and 16HBE14o-, expressing endogenous WT-CFTR; and the human airway epithelial cells JME/CF15, endogenously expressing F508del-CFTR (class II CF mutation). Login to comment
209 Experiments were performed in the presence of 1 ␮M Fsk (WT-CFTR), 10 ␮M Fsk ϩ 30 ␮M genistein (F508del- and G551D-) or 10 ␮M ϩ 250 ␮M MPB-91 (G1349D-CFTR). Login to comment
210 Compounds (Ratio a,b Determined by NMR Spectrometry) Endogenous CFTR Heterologous Expression of CFTR WT-CFTR F508del-CFTR WT-CFTR G551D-CFTR G1349D-CFTR Glibenclamide 11.7 Ϯ 1.1 ␮M 11.8 Ϯ 1.1 ␮M 14.7 Ϯ 1.3 ␮M 7.9 Ϯ 1.6 ␮M 9.0 Ϯ 1.4 ␮M CFTRinh-172 N.D. N.D. 1.2 Ϯ 0.9 ␮M N.D. N.D. 5a 93.3 Ϯ 1.3 pM 67.3 Ϯ 1.3 pM 71.0 Ϯ 1.2 pM 43.1 Ϯ 1.5 nM 72.3 Ϯ 1.1 pM 8a,b (60:40) 15.7 Ϯ 1.1 nM 8.7 Ϯ 1.2 nM 2.5 Ϯ 1.7 nM 190 Ϯ 2 nM 79.4 Ϯ 2 nM 7a,b (60:40) 2.3 Ϯ 1.5 nM 6.3 Ϯ 2.1 nM 3.4 Ϯ 1.2 nM 154 Ϯ 2 nM 7.0 Ϯ 1.1 nM 3a,b (75:25) 11.2 Ϯ 1.6 ␮M 7.0 Ϯ 1.4 ␮M 4.4 Ϯ 1.3 M 35.5 Ϯ 1.6 ␮M 9.0 Ϯ 1.8 ␮M 4a,b (60:40) 11.9 Ϯ 1.7 ␮M 6.0 Ϯ 3.3 ␮M 5.7 Ϯ 1.3 ␮M 110 Ϯ 2 ␮M 2.9 Ϯ 1.4 ␮M N.D., complete dose-response curve was not determined, but the inhibition for each cell type with 10 ␮M concentrations of inhibitors was confirmed. Login to comment
256 Responses to various adducts were also studied on CFTR chloride channels affected by class III CF mutations, G551Dand G1349D-CFTR. Login to comment
268 For G1349D-CFTR, iodide efflux was stimulated by a mixture of 10 ␮M Fsk plus CFTR activator MPB-91 (250 ␮M) because genistein failed to stimulate G1349D-CFTR as reported earlier (Melin et al., 2004). Login to comment
269 The IC50 for glibenclamide inhibition of G1349D-CFTR (9 Ϯ 1.4 ␮M; Table 1) was in this case similar to that of WT-CFTR. Login to comment
270 In contrast with G551D, adducts 3a,b, 4a,b, 5a (IC50 of 72.3 Ϯ 1.1 pM), 7a,b, and 8a,b inhibited G1349D-CFTR with affinities similar to that of WT-CFTR (Table 1). Login to comment
271 Thus, G551Dand G1349D-CFTR channels are both inhibited (although with different potencies) by the ␣-aminoazaheterocycle-methylglyoxal adducts. Login to comment
288 Discussion In this report, a new chemical class of CFTR channel inhibitors was discovered; among these are new, highly potent, water-soluble, nontoxic molecules suitable for studying CFTR with iodide efflux, microcytofluorimetry, and whole-cell patch-clamp techniques in epithelial and nonepithelial cells and for CFTR-dependent transepithelial current analysis with a Ussing chamber in mouse colon. To our knowledge, the 2Ј-deoxyadenosine derivative 5a represents the most potent inhibitor of CFTR channels with picomolar affinity in WT-, F508del- and G1349D-CFTR-expressing cells and nano- Fig. 8. Login to comment
293 B, with CHO cells overexpressing G551D-CFTR and COS-7 cells expressing G1349D-CFTR. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42. Bompadre SG, Hwang TC
Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):431-42., 2007-08-25 [PMID:17700963]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

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47 In the S.S., there are two important disease-associated mutations, G551D in NBD1 and G1349D in NBD. Login to comment
211 In contrast, the corresponding mutation in the signature sequence of NBD2, G1349D, is associated with a milder clinical phenotype[65] . Login to comment
221 In contrast, the G1349D mutation retains some ATP dependence (Fig.5), with a maximal Po approximately 10-fold lower than that of wild-type CFTR because of a reduced maximum opening rate. Login to comment
222 Although the exact mechanism for the functional defect of the G1349D mutation remains unclear, potential electrostaticrepulsionbetweennegativelychargedside chain of D1349 and bound ATP may hinder NBD dimerization (i.e., a post-binding event). Login to comment
224 Gating of G551D-CFTR and G1349D-CFTR. Login to comment
228 B: Recording of G1349D-CFTR current in an excised inside-out membrane patch. Login to comment

[hide] Scanning the cystic fibrosis transmembrane conduct... Clin Chem. 2007 Nov;53(11):1891-8. Epub 2007 Sep 21. Montgomery J, Wittwer CT, Kent JO, Zhou L
Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis.
Clin Chem. 2007 Nov;53(11):1891-8. Epub 2007 Sep 21., [PMID:17890437]

Abstract [show]
BACKGROUND: Complete gene analysis of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution DNA melting analysis is a rapid, closed-tube alternative for gene scanning and genotyping. METHODS: The 27 exons of CFTR were amplified in 37 PCR products under identical conditions. Common variants in 96 blood donors were identified in each exon by high-resolution melting on a LightScanner(R). We then performed a subsequent blinded study on 30 samples enriched for disease-causing variants, including all 23 variants recommended by the American College of Medical Genetics and 8 additional, well-characterized variants. RESULTS: We identified 22 different sequence variants in 96 blood donors, including 4 novel variants and the disease-causing p.F508del. In the blinded study, all 40 disease-causing heterozygotes (29 unique) were detected, including 1 new probable disease-causing variant (c.3500-2A>T). The number of false-positive amplicons was decreased 96% by considering the 6 most common heterozygotes. The melting patterns of most heterozygotes were unique (37 of 40 pairs within the same amplicon), the exceptions being p.F508del vs p.I507del, p.G551D vs p.R553X, and p.W1282X vs c.4002A>G. The homozygotes p.G542X, c.2789 + 5G>A, and c.3849 + 10kbC>T were directly identified, but homozygous p.F508del was not. Specific genotyping of these exceptions, as well as genotyping of the 5T allele of intron 8, was achieved by unlabeled-probe and small-amplicon melting assays. CONCLUSIONS: High-resolution DNA melting methods provide a rapid and accurate alternative for complete CFTR analysis. False positives can be decreased by considering the melting profiles of common variants.

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148 c c.1341 ϩ 18AϾC was associated with the variant p.G1349D in 1 sample. Login to comment

[hide] Mechanism of G551D-CFTR (cystic fibrosis transmemb... J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30. Bompadre SG, Li M, Hwang TC
Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.
J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30., 2008-02-29 [PMID:18167357]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). The NBDs dimerize in a head-to-tail configuration, forming two ATP binding pockets (ABP) with the ATP molecules buried at the dimer interface. Previous studies have indicated that ABP2, formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1, is the site critical for the ATP-dependent opening of CFTR. The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resulting in an open probability that is approximately 100-fold lower than that of wild-type channels. Interestingly, we found that the ATP analog N6-(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. This effect is reduced when P-ATP is applied together with ATP, suggesting a competition between ATP and P-ATP for a common binding site. Introducing mutations that lower the nucleotide binding affinity at ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. These results substantiate the idea that nucleotide binding at ABP1 stabilizes the open channel conformation. Our observation that P-ATP enhances the G551D activity by binding at ABP1 implicates that ABP1 can potentially be a target for drugs to bind and increase the channel activity.

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No. Sentence Comment
16 The importance of the signature sequence in CFTR gating is attested by the fact that mutations such as G551D and G1349D in thisregionoftheproteinareassociatedwithCF.G551D,locatedin the signature sequence of NBD1, is one of the most common CF-associated mutations (the Cystic Fibrosis Mutation Database). Login to comment

[hide] Diversity of the basic defect of homozygous CFTR m... J Med Genet. 2008 Jan;45(1):47-54. Stanke F, Ballmann M, Bronsveld I, Dork T, Gallati S, Laabs U, Derichs N, Ritzka M, Posselt HG, Harms HK, Griese M, Blau H, Mastella G, Bijman J, Veeze H, Tummler B
Diversity of the basic defect of homozygous CFTR mutation genotypes in humans.
J Med Genet. 2008 Jan;45(1):47-54., [PMID:18178635]

Abstract [show]
BACKGROUND: Knowledge of how CFTR mutations other than F508del translate into the basic defect in cystic fibrosis (CF) is scarce due to the low incidence of homozygous index cases. METHODS: 17 individuals who are homozygous for deletions, missense, stop or splice site mutations in the CFTR gene were investigated for clinical symptoms of CF and assessed in CFTR function by sweat test, nasal potential difference and intestinal current measurement. RESULTS: CFTR activity in sweat gland, upper airways and distal intestine was normal for homozygous carriers of G314E or L997F and in the range of F508del homozygotes for homozygous carriers of E92K, W1098L, R553X, R1162X, CFTRdele2(ins186) or CFTRdele2,3(21 kb). Homozygotes for M1101K, 1898+3 A-G or 3849+10 kb C-T were not consistent CF or non-CF in the three bioassays. 14 individuals exhibited some chloride conductance in the airways and/or in the intestine which was identified by the differential response to cAMP and DIDS as being caused by CFTR or at least two other chloride conductances. DISCUSSION: CFTR mutations may lead to unusual electrophysiological or clinical manifestations. In vivo and ex vivo functional assessment of CFTR function and in-depth clinical examination of the index cases are indicated to classify yet uncharacterised CFTR mutations as either disease-causing lesions, risk factors, modifiers or neutral variants.

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89 Several well characterised severe mutations occur in the evolutionarily conserved Walker (G1244E, G1249E) or dodecapeptide motifs (G551D, G1349D) of the ABC transporter CFTR.1 The missense mutants G622D23 in the regulatory domain and G314E in the fifth transmembrane region led to no clinical symptoms of CF. Login to comment

[hide] Misfolding of the cystic fibrosis transmembrane co... Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15. Cheung JC, Deber CM
Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.
Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15., 2008-02-12 [PMID:18193900]

Abstract [show]
Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.

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89 In contrast, the mutant G551D (also located in NBD1) affects mainly the function of CFTR but not its trafficking (71), while the mutant G1349D, at an equivalent position in NBD2 as G551 in NBD1, similarly affects the function of CFTR (72). Login to comment

[hide] Proteasome-dependent pharmacological rescue of cys... J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29. Norez C, Bilan F, Kitzis A, Mettey Y, Becq F
Proteasome-dependent pharmacological rescue of cystic fibrosis transmembrane conductance regulator revealed by mutation of glycine 622.
J Pharmacol Exp Ther. 2008 Apr;325(1):89-99. Epub 2008 Jan 29., [PMID:18230692]

Abstract [show]
The most common mutation (F508del) causing cystic fibrosis (CF) results in misfolding of the CF transmembrane conductance regulator (CFTR), leading to its degradation via the proteasome pathway. To study the mechanism of action of several pharmacological chaperones benzo[c]quinolizinium (MPB), we analyzed their effects on two CF mutations; F508del-CFTR and G622D-CFTR. The replacement of Gly622 by an aspartic acid (G622D) alters the trafficking and activity of the protein. G622D, similar to F508del, was functionally rescued by the glucosidase inhibitor miglustat but, unlike F508del, could not be rescued by MPB. A structure-activity relationship for F508del functional correction revealed the following profile: MPB-104-91-07-80 > 05 > 89 >> 9-hydroxyphenanthrene = phenanthrene. Coimmunoprecipitation experiments on human airway epithelial F508del/F508del CF15 cells showed that MPB did not prevent the interaction of F508del-CFTR with heat shock protein (HSP)70, HSP90, or calnexin. Functional rescue of F508del-CFTR by MPB and miglustat was abolished by brefeldin A (BFA) but potentiated by thapsigargin (TG) and geldanamycin. The proteasome inhibitor MG132 potentiated the effect of miglustat but only modestly affected that of MPB. It is noteworthy that MPB inhibited proteasome activity in F508del-CFTR-expressing cells but did not directly affect the activity of purified 20S proteasome. With the mutant G622D-CFTR, MPB did not inhibit proteasome activity, as in mock-transfected cells. Inhibition of cellular degradation machinery by MPB is not only CFTR-dependent, but it also follows similar structure-activity relationship as demonstrated by functional correction. We conclude that G622D is a partial trafficking-deficient mutant with dysfunctional chloride channel activity, and that Gly622 is part of the putative site for interaction of MPB with CFTR, protecting the channel from proteasome-mediated degradation.

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25 Several other less frequent CF mutations such as G551D or G1349D are classified as class 3 because the corresponding proteins, although correctly located at the plasma membrane, have a dysfunctional regulation (see for a recent review, MacDonald et al., 2007). Login to comment
34 In previous studies, we identified benzo[c]quinolizinium (MPB) derivatives (several chemical structures are shown in Fig. 1B together with two phenanthrene derivatives) as activators of wild-type CFTR, and G551D, G1349D, and F508del mutants (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Login to comment
234 In a previous study analyzing the effect of CFTR channel activators, we reported a structure-activity relationship of MPB compounds (Marivingt-Mounir et al., 2004), and we demonstrated that some of these derivatives were able to stimulate the channel activity of wt-, G551D-, G1349D-, G551D/ G1349D-, and F508del-CFTR (Becq et al., 1999; Dormer et al., 2001a; Marivingt-Mounir et al., 2004; Melin et al., 2004). Login to comment

[hide] Drugs and their molecular targets: an updated over... Fundam Clin Pharmacol. 2008 Feb;22(1):1-18. Landry Y, Gies JP
Drugs and their molecular targets: an updated overview.
Fundam Clin Pharmacol. 2008 Feb;22(1):1-18., [PMID:18251718]

Abstract [show]
About 330 targets bind approved drugs, 270 encoded by the human genome and 60 belonging to pathogenic organisms. A large number of druggable targets have been recently proposed from preclinical and first clinical data, but a huge reservoir of putative drug targets, possibly several thousands, remains to be explored. This overview considers the different types of ligands and their selectivity in the main superfamilies of drug targets, enzymes, membrane transporters and ion channels, and the various classes of membrane and nuclear receptors with their signalling pathway. Recently approved drugs such as monoclonal antibodies, tyrosine kinase and proteasome inhibitors, and major drugs under clinical studies are reviewed with their molecular target and therapeutic interest. The druggability of emerging targets is discussed, such as multidrug resistance transporters and cystic fibrosis transmembrane conductance regulator (CFTR), hyperpolarization-activated cyclic nucleotides-gated (HCN), cyclic nucleotide-gated (CNG) and transient receptor potential (TRP) ion channels, tumour necrosis factor (TNF) and receptor activator of NFkappaB (RANK) receptors, integrins, and orphan or recently deorphanized G-protein-coupled and nuclear receptors. Large advances have been made in the therapeutical use of recombinant cytokines and growth factors (i.e. tasonermin, TNFalpha-1a; becaplermin, platelet-derived growth factor (PDGF); dibotermin-alpha, bone morphogenetic proteins (BMP)2; anakinra, interleukin-1 receptor antagonist protein (IRAP), and in enzyme replacement therapy, i.e. algasidase (alpha-galactosidase) and laronidase (alpha-l-iduronidase). New receptor classes are emerging, e.g. membrane aminopeptidases, and novel concepts are stimulating drug research, e.g. epigenetic therapy, but the molecular target of some approved drugs, such as paracetamol and imidazolines, still need to be identified.

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92 Other mutations of CFTR, like G551D and G1349D (glycine to aspartic acid change at position 551 or 1349), cause only a gating defect. Login to comment

[hide] Evidence for direct CFTR inhibition by CFTR(inh)-1... Biochem J. 2008 Jul 1;413(1):135-42. Caci E, Caputo A, Hinzpeter A, Arous N, Fanen P, Sonawane N, Verkman AS, Ravazzolo R, Zegarra-Moran O, Galietta LJ
Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis.
Biochem J. 2008 Jul 1;413(1):135-42., 2008-07-01 [PMID:18366345]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl- channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTR(inh)-172. In the present study, we provide evidence that CFTR(inh)-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334-352 were replaced with alanine residues and the sensitivity to CFTR(inh)-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTR(inh)-172 by 20-30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTR(inh)-172 activity. The results of the present study provide evidence that CFTR(inh)-172 interacts directly with CFTR, and that Arg347 is important for the interaction.

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24 However, NBD mutants such as G551D, G1349D or P574H, do not show an altered sensitivity to CFTRinh-172 [9,12]. Login to comment

[hide] CLC-0 and CFTR: chloride channels evolved from tra... Physiol Rev. 2008 Apr;88(2):351-87. Chen TY, Hwang TC
CLC-0 and CFTR: chloride channels evolved from transporters.
Physiol Rev. 2008 Apr;88(2):351-87., [PMID:18391167]

Abstract [show]
CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level.

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846 Most notable are G551D and G1349D, two mutations located in the signature sequence of NBD1 (ABP2) and NBD2 (ABP1), respectively. Login to comment
850 On the contrary, the G1349D mutation causes a mild form of CF (271). Login to comment
853 (35) indicate that the G551D mutation completely abolishes ATP-dependent gating of CFTR (also see Ref. 329), whereas G1349D-CFTR retains some ATP dependence albeit with a Po ϳ10-fold lower than that of WT-CFTR. Login to comment

[hide] Mutations at the signature sequence of CFTR create... J Gen Physiol. 2009 Jan;133(1):69-77. Wang X, Bompadre SG, Li M, Hwang TC
Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel.
J Gen Physiol. 2009 Jan;133(1):69-77., [PMID:19114635]

Abstract [show]
The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G551D) in cystic fibrosis transmembrane conductance regulator (CFTR) results in a severe phenotype of cystic fibrosis. We previously showed that the G551D mutation completely eliminates ATP-dependent gating of the CFTR chloride channel. Here, we report that micromolar [Cd(2+)] can dramatically increase the activity of G551D-CFTR in the absence of ATP. This effect of Cd(2+) is not seen in wild-type channels or in G551A. Pretreatment of G551D-CFTR with the cysteine modification reagent 2-aminoethyl methane thiosulfonate hydrobromide protects the channel from Cd(2+) activation, suggesting an involvement of endogenous cysteine residue(s) in mediating this effect of Cd(2+). The mutants G551C, L548C, and S549C, all in the signature sequence of CFTR's NBD1, show robust response to Cd(2+). On the other hand, negligible effects of Cd(2+) were seen with T547C, Q552C, and R553C, indicating that a specific region of the signature sequence is involved in transmitting the signal of Cd(2+) binding to the gate. Collectively, these results suggest that the effect of Cd(2+) is mediated by a metal bridge formation between yet to be identified cysteine residue(s) and the engineered aspartate or cysteine in the signature sequence. We propose that the signature sequence serves as a switch that transduces the signal of ligand binding to the channel gate.

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54 This effect of Cd2+ was not seen with the wild-type (WT) channels or the corresponding mutation, G1349D, at the signature sequence of NBD2. Login to comment
70 Although Cd2+ increases the activity of G551D-CFTR, Cd2+ shows little effect on G1349D-CFTR (n = 5) (not depicted), a mutation of the corresponding glycine residue in the signature sequence of NBD2, indicating that this effect of Cd2+ is specific for the glycine-to-aspartate mutation at NBD1. Login to comment
202 G551D and G1349D, two CF-associated mutations in the signature sequence of CFTR, exhibit distinct gating defects. Login to comment
271 Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment

[hide] Gating of the CFTR Cl- channel by ATP-driven nucle... J Physiol. 2009 May 15;587(Pt 10):2151-61. Epub 2009 Mar 30. Hwang TC, Sheppard DN
Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation.
J Physiol. 2009 May 15;587(Pt 10):2151-61. Epub 2009 Mar 30., 2009-05-15 [PMID:19332488]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl- channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating.

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32 The location of the CF mutations G551D (site 2), G1349D (site 1) and F508del (surface of NBD1 opposing ICL4) are shown. Login to comment
115 The functional importance of the LSGGQ motifs in CFTR is attested by the many disease-associated mutations found in these regions of the protein (www.genet.sickkids.on.ca/cftr), most notably G551D and G1349D (Cai et al. 2006; Bompadre et al. 2007). Login to comment

[hide] State-dependent modulation of CFTR gating by pyrop... J Gen Physiol. 2009 Apr;133(4):405-19. Tsai MF, Shimizu H, Sohma Y, Li M, Hwang TC
State-dependent modulation of CFTR gating by pyrophosphate.
J Gen Physiol. 2009 Apr;133(4):405-19., [PMID:19332621]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is an adenosine triphosphate (ATP)-gated chloride channel. ATP-induced dimerization of CFTR's two nucleotide-binding domains (NBDs) has been shown to reflect the channel open state, whereas hydrolysis of ATP is associated with channel closure. Pyrophosphate (PPi), like nonhydrolytic ATP analogues, is known to lock open the CFTR channel for tens of seconds when applied with ATP. Here, we demonstrate that PPi by itself opens the CFTR channel in a Mg(2+)-dependent manner long after ATP is removed from the cytoplasmic side of excised membrane patches. However, the short-lived open state (tau approximately 1.5 s) induced by MgPPi suggests that MgPPi alone does not support a stable NBD dimer configuration. Surprisingly, MgPPi elicits long-lasting opening events (tau approximately 30 s) when administrated shortly after the closure of ATP-opened channels. These results indicate the presence of two different closed states (C(1) and C(2)) upon channel closure and a state-dependent effect of MgPPi on CFTR gating. The relative amount of channels entering MgPPi-induced long-open bursts during the ATP washout phase decreases over time, indicating a time-dependent dissipation of the closed state (C(2)) that can be locked open by MgPPi. The stability of the C(2) state is enhanced when the channel is initially opened by N(6)-phenylethyl-ATP, a high affinity ATP analogue, but attenuated by W401G mutation, which likely weakens ATP binding to NBD1, suggesting that an ATP molecule remains bound to the NBD1 site in the C(2) state. Taking advantage of the slow opening rate of Y1219G-CFTR, we are able to identify a C(2)-equivalent state (C(2)*), which exists before the channel in the C(1) state is opened by ATP. This closed state responds to MgPPi much more inefficiently than the C(2) state. Finally, we show that MgAMP-PNP exerts its effects on CFTR gating via a similar mechanism as MgPPi. The structural and functional significance of our findings is discussed.

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65 Cai et al. (2006) showed that PPi cannot potentiate G1349D, a mutation at the signature sequence of NBD2, which forms the ATP-binding pocket with the nucleotide-interacting motifs of NBD1. Login to comment
506 G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment
511 2006. Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. J. Biol. Chem. 281:1970-1977. Login to comment

[hide] Mutation-specific potency and efficacy of cystic f... J Pharmacol Exp Ther. 2009 Sep;330(3):783-91. Epub 2009 Jun 2. Caputo A, Hinzpeter A, Caci E, Pedemonte N, Arous N, Di Duca M, Zegarra-Moran O, Fanen P, Galietta LJ
Mutation-specific potency and efficacy of cystic fibrosis transmembrane conductance regulator chloride channel potentiators.
J Pharmacol Exp Ther. 2009 Sep;330(3):783-91. Epub 2009 Jun 2., [PMID:19491324]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. The mutations G551D and G1349D, which affect the nucleotide-binding domains (NBDs) of CFTR protein, reduce channel activity. This defect can be corrected pharmacologically by small molecules called potentiators. CF mutations residing in the intracellular loops (ICLs), connecting the transmembrane segments of CFTR, may also reduce channel activity. We have investigated the extent of loss of function caused by ICL mutations and the sensitivity to pharmacological stimulation. We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. The 1,4-dihydropyridine felodipine and the phenylglycine PG-01 [2-[(2-1H-indol-3-yl-acetyl)-methylamino]-N-(4-isopropylphenyl)-2-phenylac etamide] were strongly effective on the three CFTR mutants. The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Our results indicate that CFTR potentiators correct the basic defect caused by CF mutations residing in different CFTR domains. However, there are differences among potentiators, with felodipine and PG-01 having a wider pharmacological activity, and SF-01 being more mutation specific. Our observations are useful in the prioritization and development of drugs targeting the CF basic defect.

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1 The mutations G551D and G1349D, which affect the nucleotide-binding domains (NBDs) of CFTR protein, reduce channel activity. Login to comment
34 The most studied class III mutations are G551D and G1349D (Gregory et al., 1991; Bompadre et al., 2007), which alter two highly conserved glycines in the LSGGQ motif of the NBD1 and NBD2, respectively. Login to comment
36 The gating defect of ⌬F508, G551D, and G1349D can be corrected pharmacologically by small molecules called CFTR potentiators (Galietta and Moran, 2004; Verkman et al., 2006). Login to comment
39 This mechanism may counteract the effects of ⌬F508, G551D, and G1349D that instead alter NBD function. Login to comment
210 CFTR potentiators are typically effective on CF mutations localized in the NBDs, like G551D, G1349D, and ⌬F508. Login to comment
212 After detection in the primary screening, many of the active compounds have later been found to be active also on G551D and G1349D. Login to comment

[hide] Direct sensing of intracellular pH by the cystic f... J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub . Chen JH, Cai Z, Sheppard DN
Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2009 Dec 18;284(51):35495-506. Epub ., 2009-12-18 [PMID:19837660]

Abstract [show]
In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pH(i)). CFTR modulates pH(i) through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pH(i). Here, we investigate the direct effects of pH(i) on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pH(i) increased the open probability (P(o)) of wild-type CFTR, whereas alkaline pH(i) decreased P(o) and inhibited Cl(-) flow through the channel. Acidic pH(i) potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pH(i) inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pH(i) modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pH(i) dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pH(i) and inhibition by alkaline pH(i), suggesting that site 2 is a critical determinant of the pH(i) sensitivity of CFTR. The effects of pH(i) also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl(-) channel senses directly pH(i). The direct regulation of CFTR by pH(i) has important implications for the regulation of epithelial ion transport.

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6 Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Login to comment
46 These included (i) mouse mammary epithelial cells (C127 cells) expressing wild-type human CFTR, the CFTR variant ⌬R-S660A (13) or the CF mutant G1349D (14), (ii) Fischer rat thyroid epithelial cells expressing the CF mutant G551D (15), and (iii) NIH-3T3 cells expressing the CFTR construct K1250M (16). Login to comment
84 However, for G551D and G1349D, because the number of active channels in a membrane patch was unknown, we measured NPo instead of Po. Login to comment
224 Of note, the CF mutations G551D and G1349D perturb severely CFTR channel gating (14, 33). Login to comment
225 Using CFTR constructs bearing site-directed mutations, we examined the roles of the residues Lys-464, Lys-1250, Gly-551, and Gly-1349 in determining the pHi sensitivity of CFTR channel gating. Consistent with previous studies (14, 33), G1349D-CFTR and especially G551D-CFTR attenuated strongly CFTR channel gating at pHi 7.3, with brief, poorly resolved channel openings separated by very long-lasting closures (Fig. 7, A and C). Login to comment
226 Fig. 7, A-D, demonstrates that the gating behavior and, hence, NPo of G551Dand G1349D-CFTR were unaffected by pHi. Login to comment
227 Because tight dimerization of the NBDs is a prerequisite for channel opening (28) and because G551D and G1349D perturb severely channel gating (14, 33), we speculate that the effects of pHi on CFTR channel gating are dependent on the formation of an NBD1:NBD2 dimer. Login to comment
246 A, C, E, and G, representative recordings show the effects of pHi on the activity of G551D-, G1349D-, K464A-, and K1250M-CFTR Cl-channels in the presence of ATP (1 mM). Login to comment
247 Dotted lines indicate where channels are closed, and downward deflections correspond to channel openings. B, D, F, and H, effects of pHi on the NPo of G551Dand G1349D-CFTR and Po of K464A- and K1250M-CFTR. Login to comment
248 Data are means Ϯ S.E. (n ϭ 6, except G551Dand G1349D-CFTR at pHi ϭ 8.3, where n ϭ 3). Login to comment
301 Thus, the loss of pHi sensitivity by G551Dand G1349D-CFTR suggests that correct alignment of the NBD1:NBD2 dimer is required for the potentiation of CFTR channel gating by acidic pHi and inhibition by alkaline pHi. Login to comment
313 As discussed above, the failure of Hϩ ions to potentiate G551D- (site 2) and G1349D-CFTR (site 1) is likely a consequence of the profound disruption of NBD dimerization and, hence, channel gating by these constructs. Login to comment

[hide] Synthesis of 4-thiophen-2'-yl-1,4-dihydropyridines... Bioorg Med Chem. 2009 Dec 1;17(23):7894-903. Epub 2009 Oct 20. Cateni F, Zacchigna M, Pedemonte N, Galietta LJ, Mazzei MT, Fossa P, Giampieri M, Mazzei M
Synthesis of 4-thiophen-2'-yl-1,4-dihydropyridines as potentiators of the CFTR chloride channel.
Bioorg Med Chem. 2009 Dec 1;17(23):7894-903. Epub 2009 Oct 20., 2009-12-01 [PMID:19880323]

Abstract [show]
The gating of the CFTR chloride channel is altered by a group of mutations that cause cystic fibrosis. This gating defect may be corrected by small molecules called potentiators. Some 1,4-dihydropyridine (DHP) derivatives, bearing a thiophen-2-yl and a furanyl ring at the 4-position of the nucleus, were prepared and tested as CFTR potentiators. In particular, we evaluated the ability of novel DHPs to enhance the activity of the rescued DeltaF508-CFTR as measured with a functional assay based on the halide-sensitive yellow fluorescent protein. Most DHPs showed an effect comparable to or better than that of the reference compound genistein. The potency was instead significantly improved, with some compounds, such as 3g, 3h, 3n, 4a, 4b, and 4d, having a half effective concentration in the submicromolar range. CoMFA analysis gave helpful suggestions to improve the activity of DHPs.

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14 For instance, deletion of phenylalanine at position 508 (DF508), the most frequent mutation (occurring in more than 50-70% of patients),5 causes both a severe CFTR processing defect (the protein being trapped and degraded at the endoplasmic [ER] level) and a decrease of its channel activity.6-8 Other mutations, like G551D and G1349D, produce only a gating defect. Login to comment
18 In this regard, we have found that the 1,4-dihydropyridines (DHPs) used to treat hypertension are acting as potentiators and so are able to stimulate the activity of some CFTR mutants (in particular, G551D, G1349D and rescued DF508-CFTR).14,15 Antihypertensive DHPs act by blocking L-type voltage-dependent Ca2+ channels (L-VDCC) and therefore cause the relaxation of arterial 0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. Login to comment

[hide] Cystic fibrosis: exploiting its genetic basis in t... Pharmacol Ther. 2010 Feb;125(2):219-29. Epub 2009 Nov 10. Kreindler JL
Cystic fibrosis: exploiting its genetic basis in the hunt for new therapies.
Pharmacol Ther. 2010 Feb;125(2):219-29. Epub 2009 Nov 10., [PMID:19903491]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in epithelial cells throughout the body. In the lungs, absence or dysfunction of CFTR results in altered epithelial salt and water transport eventuating in impaired mucociliary clearance, chronic infection and inflammation, and tissue damage. CF lung disease is the major cause of morbidity and mortality in CF despite the many therapies aimed at reducing it. However, recent technological advances combined with two decades of research driven by the discovery of the CFTR gene have resulted in the development and clinical testing of novel therapies aimed at the principal underlying defect in CF, thereby ushering in a new age of therapy for CF.

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555 In Cos-7 cells, G551D CFTR responds differently to genistein than does G551D/G1349D CFTR, suggesting that both NBD sites play a role in the activity of genistein (Melin et al., 2004). Login to comment

[hide] Inhibition of protein kinase CK2 closes the CFTR C... Cell Physiol Biochem. 2009;24(5-6):347-60. Epub 2009 Nov 4. Treharne KJ, Xu Z, Chen JH, Best OG, Cassidy DM, Gruenert DC, Hegyi P, Gray MA, Sheppard DN, Kunzelmann K, Mehta A
Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR.
Cell Physiol Biochem. 2009;24(5-6):347-60. Epub 2009 Nov 4., [PMID:19910675]

Abstract [show]
BACKGROUND: Deletion of phenylalanine-508 (DeltaF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between DeltaF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2. METHODS: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies. RESULTS: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl(-) transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not DeltaF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and DeltaF508CFTR expressing oocytes, only DeltaF508-CFTR Cl(-) currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the DeltaF508-equivalent peptide had no effect. CONCLUSIONS: CK2 controls wtCFTR, but not DeltaF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by DeltaF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR.

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345 J Biol Chem 2000;275:36632-36636. 35 Cai Z, Taddei A, Sheppard DN: Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment

[hide] Influence of cell background on pharmacological re... Am J Physiol Cell Physiol. 2010 Apr;298(4):C866-74. Epub 2010 Jan 6. Pedemonte N, Tomati V, Sondo E, Galietta LJ
Influence of cell background on pharmacological rescue of mutant CFTR.
Am J Physiol Cell Physiol. 2010 Apr;298(4):C866-74. Epub 2010 Jan 6., [PMID:20053923]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue.

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17 Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. Login to comment
31 Class 3 mutations, such as G1349D and G551D (2, 11), have normal trafficking but are affected by a gating defect more severe than that of F508del. Login to comment
32 G551D and G1349D alter two highly conserved glycines in NBD1 and NBD2, respectively. Login to comment
53 In brief, FRT cells were first stably transfected with the plasmid pTRACER (Invitrogen) containing the cDNA encoding CFTR (wild type or mutant F508del, G1349D, or G551D) and selected in Zeocin (0.6 mg/ml). Login to comment
80 FRT cells expressing mutant G1349D or G551D were grown at 37°C (90% humidity; 5% CO2) for 18-24 h. Login to comment
89 Potentiator activity on G551Dand G1349D-CFTR. Login to comment
90 A: original traces recorded in FRT cells expressing G1349D-CFTR (top) and G551D-CFTR (bottom), showing quenching of cellular YFP fluorescence by I- addition after stimulation with forskolin (20 ␮M) alone or forskolin ϩ indicated compounds (20 ␮M and 45 ␮M for G1349D and G551D, respectively). Login to comment
91 B: dose-response analysis of indicated compounds in G1349D-CFTR (top) and G551D-CFTR (bottom) cells (means Ϯ SE, n ϭ 6). Login to comment
92 C: maximal effect obtained from dose response of potentiators in G1349D-CFTR (left) and G551D-CFTR (right) cells, normalized to genistein effect (means Ϯ SE, n ϭ 9). Login to comment
94 D: correlation between potentiator activity on G1349D-CFTR and G551D-CFTR. Login to comment
95 Each symbol represents the activity of a single potentiator on G1349D (left)- and G551D (right)- vs. F508del-CFTR. Login to comment
97 Note that the concentrations needed to stimulate the G551D mutant are always 5-10 times higher than those effective on F508del or G1349D. Login to comment
98 Gray lines indicate correlation lines (R ϭ 0.96 for G1349D and R ϭ 0.95 for G551D). Login to comment
157 Potentiators were also tested on FRT cells expressing the G1349D and the G551D mutants with the YFP assay (Fig. 3). Login to comment
159 In contrast, the gating defect of the G1349D mutant was less severe, allowing a significant cAMP-activated anion transport. Login to comment
160 CFTR activity in G551D and G1349D cells was strongly stimulated in the presence of potentiators (Fig. 3A). Login to comment
165 Interestingly, the order of potency for active potentiators in G551D and G1349D correlated well with the order of potency in F508del (Fig. 3D). Login to comment
166 However, the Ka values for G551D were consistently 5-10 times higher than those for G1349D and F508del. Login to comment
240 Potentiators were also tested on two classical CF mutations belonging to class 3 (gating defect), namely, G551D and G1349D. Login to comment
241 Such mutations are particularly interesting because they cause a drastic reduction in CFTR activity (with G551D being the most severe mutation) and because they involve two highly conserved glycine residues in the first and second NBDs, respectively. Login to comment
244 Other potentiators were also less effective in activating G551D, such as the sulfonamide P3. Login to comment
245 In general, the order of potency of the active potentiators was similar for G551D, G1349D, and F508del. Login to comment
248 Our findings suggest that all potentiators having activity on G551D, G1349D, and F508del (P1-P7 and DHPs) act with the same mechanism of action, possibly through binding to the same site on CFTR protein. Login to comment
13 Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. Login to comment
27 Class 3 mutations, such as G1349D and G551D (2, 11), have normal trafficking but are affected by a gating defect more severe than that of F508del. Login to comment
28 G551D and G1349D alter two highly conserved glycines in NBD1 and NBD2, respectively. Login to comment
49 In brief, FRT cells were first stably transfected with the plasmid pTRACER (Invitrogen) containing the cDNA encoding CFTR (wild type or mutant F508del, G1349D, or G551D) and selected in Zeocin (0.6 mg/ml). Login to comment
76 FRT cells expressing mutant G1349D or G551D were grown at 37°C (90% humidity; 5% CO2) for 18-24 h. Login to comment
85 Potentiator activity on G551D- and G1349D-CFTR. Login to comment
86 A: original traces recorded in FRT cells expressing G1349D-CFTR (top) and G551D-CFTR (bottom), showing quenching of cellular YFP fluorescence by I- addition after stimulation with forskolin (20 ␮M) alone or forskolin ϩ indicated compounds (20 ␮M and 45 ␮M for G1349D and G551D, respectively). Login to comment
87 B: dose-response analysis of indicated compounds in G1349D-CFTR (top) and G551D-CFTR (bottom) cells (means Ϯ SE, n ϭ 6). Login to comment
88 C: maximal effect obtained from dose response of potentiators in G1349D-CFTR (left) and G551D-CFTR (right) cells, normalized to genistein effect (means Ϯ SE, n ϭ 9). Login to comment
93 Note that the concentrations needed to stimulate the G551D mutant are always 5-10 times higher than those effective on F508del or G1349D. Login to comment
153 Potentiators were also tested on FRT cells expressing the G1349D and the G551D mutants with the YFP assay (Fig. 3). Login to comment
155 In contrast, the gating defect of the G1349D mutant was less severe, allowing a significant cAMP-activated anion transport. Login to comment
156 CFTR activity in G551D and G1349D cells was strongly stimulated in the presence of potentiators (Fig. 3A). Login to comment
161 Interestingly, the order of potency for active potentiators in G551D and G1349D correlated well with the order of potency in F508del (Fig. 3D). Login to comment
162 However, the Ka values for G551D were consistently 5-10 times higher than those for G1349D and F508del. Login to comment
236 Potentiators were also tested on two classical CF mutations belonging to class 3 (gating defect), namely, G551D and G1349D. Login to comment

[hide] ATP-independent CFTR channel gating and allosteric... Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3. Wang W, Wu J, Bernard K, Li G, Wang G, Bevensee MO, Kirk KL
ATP-independent CFTR channel gating and allosteric modulation by phosphorylation.
Proc Natl Acad Sci U S A. 2010 Feb 23;107(8):3888-93. Epub 2010 Feb 3., 2010-02-23 [PMID:20133716]

Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, an ATP binding cassette (ABC) transporter. CFTR gating is linked to ATP binding and dimerization of its two nucleotide binding domains (NBDs). Channel activation also requires phosphorylation of the R domain by poorly understood mechanisms. Unlike conventional ligand-gated channels, CFTR is an ATPase for which ligand (ATP) release typically involves nucleotide hydrolysis. The extent to which CFTR gating conforms to classic allosteric schemes of ligand activation is unclear. Here, we describe point mutations in the CFTR cytosolic loops that markedly increase ATP-independent (constitutive) channel activity. This finding is consistent with an allosteric gating mechanism in which ligand shifts the equilibrium between inactive and active states but is not essential for channel opening. Constitutive mutations mapped to the putative symmetry axis of CFTR based on the crystal structures of related ABC transporters, a common theme for activating mutations in ligand-gated channels. Furthermore, the ATP sensitivity of channel activation was strongly enhanced by these constitutive mutations, as predicted for an allosteric mechanism (reciprocity between protein activation and ligand occupancy). Introducing constitutive mutations into CFTR channels that cannot open in response to ATP (i.e., the G551D CF mutant and an NBD2-deletion mutant) substantially rescued their activities. Importantly, constitutive mutants that opened without ATP or NBD2 still required R domain phosphorylation for optimal activity. Our results confirm that (i) CFTR gating exhibits features of protein allostery that are shared with conventional ligand-gated channels and (ii) the R domain modulates CFTR activity independent of ATP-induced NBD dimerization.

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252 Bompadre SG, Sohma Y, Li M, Hwang T-C (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment
241 Bompadre SG, Sohma Y, Li M, Hwang T-C (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] Stable ATP binding mediated by a partial NBD dimer... J Gen Physiol. 2010 May;135(5):399-414. Tsai MF, Li M, Hwang TC
Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.
J Gen Physiol. 2010 May;135(5):399-414., [PMID:20421370]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.

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402 G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] Identification of the second CFTR mutation in pati... Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26. Giuliani R, Antonucci I, Torrente I, Grammatico P, Palka G, Stuppia L
Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.
Asian J Androl. 2010 Nov;12(6):819-26. Epub 2010 Jul 26., [PMID:20657600]

Abstract [show]
Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.

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58 INNO-LiPA CFTR19 INNO-LiPA CFTR17 INNO-LiPA CFTR Italian regional [delta]F508 621+1G>T 1259insA G542X 3849+10kbC>T 4016insT N1303K 2183AA>G 4382delA W1282X 394delTT 852del22 G551D 2789+5G> A R1162X D579G 1717-1G>A 3659delC G1244E R553X R117H G1349D CFTRdele2,3 (21 kb) R334W I502T [delta]I507 R347P L1065P 711+1G>T G85E R1158X 3272-26A>G 3905insT 1078delT T338I R560T A455E S549R(A>C) 1898+1G>A S1251N 2143delA 711+5G>A 991del5 I148T E60X D1152H 3199del6 3120+1G>A 2184delA 1898+3A>G, R1070Q Q552X Poli-T tract variations R1066H R347H 621+3A>G R334Q E217G Abbreviation: CFTR, cystic fibrosis transmembrane conductance regulator. Login to comment

[hide] Dual roles of the sixth transmembrane segment of t... J Gen Physiol. 2010 Sep;136(3):293-309. Bai Y, Li M, Hwang TC
Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation.
J Gen Physiol. 2010 Sep;136(3):293-309., [PMID:20805575]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily that functions as a chloride channel. Previous work has suggested that the external side of the sixth transmembrane segment (TM6) plays an important role in governing chloride permeation, but the function of the internal side remains relatively obscure. Here, on a cysless background, we performed cysteine-scanning mutagenesis and modification to screen the entire TM6 with intracellularly applied thiol-specific methanethiosulfonate reagents. Single-channel amplitude was reduced in seven cysteine-substituted mutants, suggesting a role of these residues in maintaining the pore structure for normal ion permeation. The reactivity pattern of differently charged reagents suggests that the cytoplasmic part of TM6 assumes a secondary structure of an alpha helix, and that reactive sites (341, 344, 345, 348, 352, and 353) reside in two neighboring faces of the helix. Although, as expected, modification by negatively charged reagents inhibits anion permeation, interestingly, modification by positively charged reagents of cysteine thiolates on one face (344, 348, and 352) of the helix affects gating. For I344C and M348C, the open time was prolonged and the closed time was shortened after modification, suggesting that depositions of positive charges at these positions stabilize the open state but destabilize the closed state. For R352C, which exhibited reduced single-channel amplitude, modifications by two positively charged reagents with different chemical properties completely restored the single-channel amplitude but had distinct effects on both the open time and the closed time. These results corroborate the idea that a helix rotation of TM6, which has been proposed to be part of the molecular motions during transport cycles in other ABC transporters, is associated with gating of the CFTR pore.

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420 G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] Mutations that permit residual CFTR function delay... Respir Res. 2010 Oct 8;11:140. Green DM, McDougal KE, Blackman SM, Sosnay PR, Henderson LB, Naughton KM, Collaco JM, Cutting GR
Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients.
Respir Res. 2010 Oct 8;11:140., [PMID:20932301]

Abstract [show]
BACKGROUND: Lung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF. METHODS: Lung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function CFTR mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories. RESULTS: Subjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile). CONCLUSIONS: Residual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.

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74 For Pa, the hazard ratio Table 1 Classification of CFTR alleles Category Mutation Specific mutations Class I Defective Protein Synthesis (nonsense, frameshift, aberrant splicing) 1078delT, 1154 insTC, 1525-2A > G, 1717-1G > A, 1898+1G > A, 2184delA, 2184 insA, 3007delG, 3120+1G > A, 3659delC, 3876delA, 3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+1G > T, 441delA, 556delA, 621+1G > T, 621-1G > T, 711+1G > T, 875+1G > C, E1104X, E585X, E60X, E822X, G542X, G551D/R553X, Q493X, Q552X, Q814X, R1066C, R1162X, R553X, V520F, W1282X, Y1092X Class II Abnormal Processing and Trafficking A559T, D979A, ΔF508, ΔI507, G480C, G85E, N1303K, S549I, S549N, S549R Class III Defective Channel Regulation/Gating G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, S549 (R75Q) Class IV Decreased Channel Conductance A800G, D1152H, D1154G, D614G, delM1140, E822K, G314E, G576A, G622D, G85E, H620Q, I1139V, I1234V, L1335P, M1137V, P67L, R117C, R117P, R117H, R334W, R347H, R347P, R347P/ R347H, R792G, S1251N, V232D Class V Reduced Synthesis and/or Trafficking 2789+5G > A, 3120G > A, 3272-26A > G, 3849+10kbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, P574H was increased 3 fold for those with 'Minimal` function when compared to those with 'Residual` function. Login to comment

[hide] Modulation of cystic fibrosis transmembrane conduc... J Biol Chem. 2010 Dec 31;285(53):41591-6. Epub 2010 Oct 25. Melani R, Tomati V, Galietta LJ, Zegarra-Moran O
Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH.
J Biol Chem. 2010 Dec 31;285(53):41591-6. Epub 2010 Oct 25., 2010-12-31 [PMID:20974851]

Abstract [show]
Potentiators are molecules that increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Some potentiators can also inhibit CFTR at higher concentrations. The activating binding site is thought to be located at the interface of the dimer formed by the two nucleotide-binding domains. We have hypothesized that if binding of potentiators involves titratable residues forming salt bridges, then modifications of cytosolic pH (pH(i)) would alter the binding affinity. Here, we analyzed the effect of pH(i) on CFTR activation and on the binding of genistein, a well known CFTR potentiator. We found that pH(i) does modify CFTR maximum current (I(m)) and half-activation concentration (K(d)): I(m) = 127.7, 185.5, and 231.8 muA/cm(2) and K(d) = 32.7, 56.6 and 71.9 mum at pH 6, 7.35, and 8, respectively. We also found that the genistein apparent dissociation constant for activation (K(a)) increased at alkaline pH(i), near cysteine pK (K(a) = 1.83, 1.81 and 4.99 mum at pH(i) 6, 7.35, and 8, respectively), suggesting the involvement of cysteines in the binding site. Mutations of cysteine residues predicted to be within (Cys-491) or outside (Cys-1344) the potentiator-binding site showed that Cys-491 is responsible for the sensitivity of potentiator binding to alkaline pH(i). Effects of pH(i) on inhibition by high genistein doses were also analyzed. Our results extend previous data about multiple effects of pH(i) on CFTR activity and demonstrate that binding of potentiators involves salt bridge formation with amino acids of nucleotide-binding domain 1.

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23 First, CFTR carrying NBD mutations, such as G551D and G1349D, exhibit a lower affinity for several potentiators, indicating that mutations are close to the binding site (21-23). Login to comment

[hide] On the mechanism of CFTR inhibition by a thiazolid... J Gen Physiol. 2010 Dec;136(6):659-71. Epub 2010 Nov 15. Kopeikin Z, Sohma Y, Li M, Hwang TC
On the mechanism of CFTR inhibition by a thiazolidinone derivative.
J Gen Physiol. 2010 Dec;136(6):659-71. Epub 2010 Nov 15., [PMID:21078867]

Abstract [show]
The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-th iazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.

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66 However, it is strange that the degree of inhibition for G551D and G1349D was of the same order of magnitude as that of wild-type (WT)-CFTR, despite a very different closed time among these three channels (Bompadre et al., 2007). Login to comment
386 G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment
391 Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel. Login to comment

[hide] Dual activity of aminoarylthiazoles on the traffic... J Biol Chem. 2011 Apr 29;286(17):15215-26. Epub 2011 Mar 7. Pedemonte N, Tomati V, Sondo E, Caci E, Millo E, Armirotti A, Damonte G, Zegarra-Moran O, Galietta LJ
Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations.
J Biol Chem. 2011 Apr 29;286(17):15215-26. Epub 2011 Mar 7., 2011-04-29 [PMID:21383017]

Abstract [show]
A large fraction of mutations causing cystic fibrosis impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by causing reduced channel activity (gating defect) and/or impaired exit from the endoplasmic reticulum (trafficking defect). Such defects need to be treated with separate pharmacological compounds termed potentiators and correctors, respectively. Here, we report the characterization of aminoarylthiazoles (AATs) as compounds having dual activity. Cells expressing mutant CFTR were studied with functional assays (fluorescence-based halide transport and short circuit current measurements) to assess the effect of acute and chronic treatment with compounds. We found that AATs are effective on F508del, the most frequent cystic fibrosis mutation, which is associated with both a gating and a trafficking defect. AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Evaluation of a panel of AAT analogs identified EN277I as the most effective compound. Incubation of cells expressing mutant CFTR with EN277I caused a strong stimulation of channel activity as demonstrated by single channel recordings. Compounds with dual activity such as AATs may be useful for the development of effective drugs for the treatment of cystic fibrosis.

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4 AATs are also effective on mutations like G1349D and G551D, which cause only a gating defect. Login to comment
14 Conversely, class III mutations (like G551D and G1349D) do not impair protein trafficking but severely decrease CFTR channel opening in response to cAMP elevation. Login to comment
21 In other words, maximal stimulation of mutant CFTR with a cAMP agonist evokes only a fraction (20-40% for F508del, less than 5-10% for G551D and G1349D) of the total activity obtained by also adding a potentiator (10, 11, 17-19). Login to comment
47 EXPERIMENTAL PROCEDURES Cell Culture-Fischer rat thyroid (FRT) cells were stably transfected with F508del, G551D, or G1349D-CFTR (12). Login to comment
100 Patch Clamp Experiments-Single channel analysis was done on FRT cells stably expressing CFTR with G1349D mutation in cell-attached and inside-out configurations. Login to comment
213 Therefore, we tested corr-2b and EN277I on FRT cells expressing G1349D-CFTR or G551D-CFTR mutant, using the HS-YFP assay and short circuit current recordings (Fig. 5). Login to comment
214 On G1349D-CFTR cells, long term treatment with AATs or corr-4a did not change QRTOT (Fig. 5A, top panel). Login to comment
228 Using the HS-YFP assay, we compared the concentrations of EN277I and genistein that are effective in improving the relative forskolin response of F508del-, G1349D-, and G551D-CFTR mutants (Fig. 5C). Login to comment
231 Indeed the Kd values for genistein were: 11.8 Ϯ 1.5 ␮M for F508del; 9.7 Ϯ 0.7 ␮M for G1349D; and 62.4 Ϯ 5.3 ␮M for G551D. Login to comment
235 The Kd values were as follows: 19.2 Ϯ 0.8 and 20.1 Ϯ 2.2 ␮M for F508del (correction of trafficking and gating defects, respectively); 15.8 Ϯ 1.1 ␮M for G1349D; and 25.1 Ϯ 1.9 ␮M for G551D. Login to comment
237 The ability of AATs to correct the gating defect of class 3 mutants after long term treatment was confirmed performing short circuit current recordings on FRT cells expressing G1349D- or G551D-CFTR (Fig. 5, D and E). Login to comment
241 A and B, the bar graphs show the total CFTR activity elicited by forskolin (20 ␮M) plus genistein (50 ␮M for G1349D and 200 ␮M for G551D) (QRTOT, top panels) and the relative activity stimulated by forskolin alone (QRFSK/QRTOT, bottom panels) in FRT cells expressing G1349D-CFTR (A) or G551D-CFTR (B) treated for 24h at 37 °C with the indicated compounds (means Ϯ S.E., n ϭ 8). Login to comment
247 D and E, representative traces of chloride current measured in Ussing chambers on G1349D-CFTR (D) and G551D-CFTR (E) FRT epithelia incubated for 24 h at 37 °C in the absence or presence of EN277I (30 ␮M) or corr-2b (50 ␮M). Login to comment
248 The concentrations were: forskolin, 20 ␮M; genistein, 50 ␮M for G1349D and 200 ␮M for G551D; CFTRinh-172 (inh-172), 10 ␮M. Login to comment
250 Incubation of G1349D cells for 24 h with EN277I or corr-2b did not change the absolute ITOT value, as compared with control condition but enhanced the percentage of the total current activated by forskolin alone, with IFSK/ITOT increasing from Ͻ10% to ϳ50%, as shown in Fig. 5D (control, IFSK ϭ 8.0 Ϯ 1.6 ␮A; corr-2b, IFSK ϭ 35.5 Ϯ 1.5 ␮A, p Ͻ 0.01; EN277I, IFSK ϭ 31.5 Ϯ 1.8 ␮A, p Ͻ 0.01; control, ITOT ϭ 70 Ϯ 6 ␮A; corr-2b, ITOT ϭ 69 Ϯ 8 ␮A, not significant; EN277I, ITOT ϭ 72 Ϯ 7 ␮A, not significant). Login to comment
252 The activity of EN277I on G1349D-CFTR expressed in FRT cells was examined in patch clamp experiments, in cell-attached and in inside-out patch configurations following acute or chronic exposure. Login to comment
260 We examined also the effect of chronic treatment of G1349D-CFTR cells with EN277I (Fig. 7). Login to comment
262 Cells treated with the vehicle alone showed low G1349D-CFTR activity, with rare openings separated by long channel closings. Login to comment
269 Patch clamp analysis of the acute effect of compound EN277I on G1349D-CFTR. Login to comment
295 We have also tested AATs on pure class III mutants, namely G1349D and G551D, the latter being the one with the most severe gating defect. Login to comment
296 Interestingly, we found that dual acting AATs also enhance the fraction of G1349D and G551D activity that is evoked by cAMP, in the absence of the potentiator. Login to comment
297 The extent of gating enhancement is larger for G1349D relative to the other mutant. Login to comment
318 Patch clamp analysis of the chronic effect of compound EN277I on G1349D-CFTR. Login to comment

[hide] Mutant cycles at CFTR's non-canonical ATP-binding ... J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16. Szollosi A, Muallem DR, Csanady L, Vergani P
Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.
J Gen Physiol. 2011 Jun;137(6):549-62. doi: 10.1085/jgp.201110608. Epub 2011 May 16., [PMID:21576373]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains closed throughout the gating cycle.

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289 Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. J. Biol. Chem. 281:1970-1977. doi:10 .1074/jbc.M510576200 Carson, M.R., M.C. Winter, S.M. Travis, and M.J. Welsh. 1995. Login to comment

[hide] High-throughput screening of libraries of compound... Methods Mol Biol. 2011;741:13-21. Pedemonte N, Zegarra-Moran O, Galietta LJ
High-throughput screening of libraries of compounds to identify CFTR modulators.
Methods Mol Biol. 2011;741:13-21., [PMID:21594775]

Abstract [show]
Small molecules acting as selective activators (potentiators), inhibitors, or "correctors" of the CFTR chloride channel represent candidate drugs for various pathological conditions including cystic fibrosis and secretory diarrhea. The identification of CFTR pharmacological modulators may be achieved by screening highly diverse synthetic or natural compound libraries using high-throughput methods. A convenient assay for CFTR function is based on the halide sensitivity of the yellow fluorescent protein (YFP). CFTR activity can be simply assessed by measuring the rate of YFP signal decrease caused by iodide influx. This assay can be automated to test thousands of compounds per day.

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8 In cystic fibrosis (CF), mutations affecting the CFTR gene cause a large variety of defects including altered CFTR channel gating (class III mutations such as G551D and G1349D) or impaired CFTR protein maturation (class II mutations such as F508del). Login to comment
135 3.5.3. Conditions for the Screening of Potentiators on G551Dand G1349D-CFTR Cells The conditions are identical to those used to test potentiators on F508del-CFTR cells except that the step of incubation at low temperature is omitted. Login to comment

[hide] Pharmacological therapy for cystic fibrosis: from ... J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45. Becq F, Mall MA, Sheppard DN, Conese M, Zegarra-Moran O
Pharmacological therapy for cystic fibrosis: from bench to bedside.
J Cyst Fibros. 2011 Jun;10 Suppl 2:S129-45., [PMID:21658632]

Abstract [show]
With knowledge of the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR), its physiological role and dysfunction in cystic fibrosis (CF), therapeutic strategies are now being developed that target the root cause of CF rather than disease symptoms. Here, we review progress towards the development of rational new therapies for CF. We highlight the discovery of small molecules that rescue the cell surface expression and defective channel gating of CF mutants, termed CFTR correctors and CFTR potentiators, respectively. We draw attention to alternative approaches to restore epithelial ion transport to CF epithelia, including inhibitors of the epithelial Na(+) channel (ENaC) and activators of the Ca(2+)-activated Cl(-) channel TMEM16A. The expertise required to translate small molecules identified in the laboratory to drugs for CF patients depends on our ability to coordinate drug development at an international level and our ability to provide pertinent biological information using suitable disease models.

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237 [136] COS-7 G551D, G1349D, G551D/G1349D Electrophysiology, iodide efflux Study explores the contribution of G551 and G1349 to CFTR modulation (potentiation and inhibition) by phloxine B. Login to comment
238 [137] C127, FRT WT, G551D, G1349D Electrophysiology Phloxine B has distinct effects on G551D and G1349D-CFTR, suggesting that drug therapy for CF is mutation-specific. Login to comment
243 [141] COS-7, HEK293, FRT G551D, G1349D, E193K, G970R YFP cell-based assay, electrophysiology Study demonstrates that potentiators are active on mutations residing in different CFTR domains and that potencies are mutation-specific. Login to comment
246 [144] FRT G551D, G1349D, temperature-corrected F508del YFP cell-based assay, electrophysiology Structure-activity relationships for CFTR potentiation and Ca2+-channel inhibition leading to identification of compounds that more potently potentiate CFTR than block Ca2+-channels. Login to comment

[hide] Validation of double gradient denaturing gradient ... Clin Chem. 1999 Jan;45(1):35-40. Cremonesi L, Carrera P, Fumagalli A, Lucchiari S, Cardillo E, Ferrari M, Righetti SC, Zunino F, Righetti PG, Gelfi C
Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis.
Clin Chem. 1999 Jan;45(1):35-40., [PMID:9895335]

Abstract [show]
Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes--the cystic fibrosis transmembrane conductance regulator (CFTR), the beta-globin, and the p53 genes--to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.

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31 Mutations and polymorphisms analyzed in the CFTR gene. Position Denaturant gradient Mutation Exon 1 40-90% 125G/Ca,b M1V (A3G at 133) 175insT 182delT Exon 3 10-60% W57G (T3G at 301) 356G/Aa G85E (G3A at 386) Exon 4 20-70% R117H (G3A at 482) 541delC 621ϩ1G3T I148T (T3C at 575) Exon 5 20-70% E193K (G3A at 709) Intron 5 20-70% 711ϩ3A3G Exon 7 20-70% 1078delT R334W (C3T at 1132) T338I (C3T at 1145) R347P (G3C at 1172)b R347H (G3A at 1172) R352Q (G3A at 1187) Exon 10 20-70% M470V (1540A/G)a ⌬F508 (del 3 bp at 1652) Intron 10 10-60% 1717-1G3A Exon 11 10-60% G542X (G3T at 1756) 1784delG R553X (C3T at 1789) Exon 12 10-60% D579G (A3G at 1868) E585X (G3T at 1885) Intron 12 10-60% 1898ϩ3A3G Exon 13 30-80% 2183AA3G E730X (G3T at 2320) L732X (T3G at 2327) 2347delG Exon 14a 10-60% T854T (2694T/G)a V868V (2736G/A)a Intron 14b 30-80% 2789ϩ5G3A Exon 15 20-70% M952I (G3C at 2988)b Exon 17a 20-70% L997F (G3C at 3123)b Exon 17b 20-70% F1052V (T3G at 3286) R1066C (C3T at 3328) R1066H (G3A at 3329) A1067T (G3A at 3331) Exon 18 20-70% D1152H (G3C at 3586)b Exon 19 30-80% R1158X (C3T at 3604) Exon 20 20-70% S1251N (G3A at 3384) W1282X (G3A at 3978) Exon 21 20-70% N1303K (C3G at 4041)b Exon 22 30-80% G1349D (G3A at 4178) 4382delA Exon 24 30-80% Y1424Y (4404C/T)a a Polymorphism. Login to comment

[hide] Adenosine and its nucleotides activate wild-type a... Am J Physiol. 1999 Feb;276(2 Pt 1):C361-9. Clancy JP, Ruiz FE, Sorscher EJ
Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2B receptor-coupled pathway.
Am J Physiol. 1999 Feb;276(2 Pt 1):C361-9., [PMID:9950763]

Abstract [show]
ATP and its metabolites stimulate Cl- secretion in human epithelium in vitro and in vivo. The specific purinergic receptor subtypes that govern these effects have been difficult to separate, in part due to multiple parallel pathways for Cl- secretion in respiratory and intestinal epithelia. In a simplified model using COS-7 cells, we demonstrate acquisition of an ATP-, ADP-, AMP-, and adenosine (ADO)-regulated halide permeability specifically following expression of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). This halide permeability is blocked by the P1 purinergic receptor antagonist 8-phenyl theophylline, sensitive to the protein kinase A inhibitor H-89, and associated with a modest, dose-dependent increase in cellular cAMP concentration. Phorbol esters poorly activate halide permeability compared with ADO, and ADO-stimulated efflux was not affected by treatment with the protein kinase C inhibitor bisindolylmaleimide I. The A2 ADO receptor (AR) agonists 5'-N-ethylcarboxamide adenosine and ADO were strong activators, whereas the A1 AR agonist R-phenylisopropyladenosine failed to activate halide permeability. Metabolic conversion of ADO nucleotides by surface ecto-5'-nucleotidase to more active (less phosphorylated) forms contributes to anion transport activation in these cells. Immunoprecipitation with anti-A2B AR antibody identified a 31-kDa protein in both COS-7 and human bronchial epithelial cells. Together, these findings indicate that ADO and its nucleotides are capable of activating wtCFTR-dependent halide permeability through A2B AR and that this AR subtype is present in human bronchial epithelium. We also present data showing that this pathway can activate clinically significant mutant CFTR molecules such as R117H.

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251 For example, additional experiments in COS-7 cells have recently allowed us to identify two other surface-localized CFTR mutants (A455E and G1349D) that can functionally couple to A2B AR activation (11). Login to comment

[hide] A potentiator induces conformational changes on th... Cell Mol Life Sci. 2012 Nov;69(21):3701-13. doi: 10.1007/s00018-012-1049-7. Epub 2012 Jul 3. Galfre E, Galeno L, Moran O
A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution.
Cell Mol Life Sci. 2012 Nov;69(21):3701-13. doi: 10.1007/s00018-012-1049-7. Epub 2012 Jul 3., [PMID:22752155]

Abstract [show]
Nucleotide binding domains (NBD1 and NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, are responsible for controlling the gating of the chloride channel and are the putative binding sites for several candidate drugs in the disease treatment. We studied the effects of the application of 2-pyrimidin-7,8-benzoflavone (PBF), a strong potentiator of the CFTR, on the properties of recombinant and equimolar NBD1/NBD2 mixture in solution. The results indicate that the potentiator induces significant conformational changes of the NBD1/NBD2 dimer in solution. The potentiator does not modify the ATP binding constant, but reduces the ATP hydrolysis activity of the NBD1/NBD2 mixture. The intrinsic fluorescence and the guanidinium denaturation measurements indicate that the potentiator induces different conformational changes on the NBD1/NBD2 mixture in the presence and absence of ATP. It was confirmed from small-angle X-ray scattering experiments that, in absence of ATP, the NBD1/NBD2 dimer was disrupted by the potentiator, but in the presence of 2 mM ATP, the two NBDs kept dimerised, and a major change in the size and the shape of the structure was observed. We propose that these conformational changes could modify the NBDs-intracellular loop interaction in a way that would facilitate the open state of the channel.

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36 This hypothesis is supported by the observation that mutations in conserved residues of the NBDs, such as G551D and G1349D, exhibit a shift in the affinity for potentiators [14, 23-25]. Login to comment
38 With the aim of identifying the activating binding site of potentiators, we modelled the NBD dimer [25], and compared the theoretical binding-free energy of several compounds docked on the model, with the experimental binding-free energy obtained from dissociation constants from wild-type, G551D, and G1349D proteins. Login to comment

[hide] Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate ... ChemMedChem. 2012 Oct;7(10):1799-807. doi: 10.1002/cmdc.201200311. Epub 2012 Aug 27. Giampieri M, Vanthuyne N, Nieddu E, Mazzei MT, Anzaldi M, Pedemonte N, Galietta LJ, Roussel C, Mazzei M
Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).
ChemMedChem. 2012 Oct;7(10):1799-807. doi: 10.1002/cmdc.201200311. Epub 2012 Aug 27., [PMID:22927224]

Abstract [show]
Some of the genetic mutations that cause cystic fibrosis (CF) impair the gating of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) ion channel. This defect can be corrected with pharmacological tools (potentiators) that belong to various chemical families, including the 1,4-dihydropyridines (DHPs). A small set of asymmetric 4-aryl-DHPs was synthesized, and each racemic couple was tested in a functional assay carried out on cells expressing the G1349D, DeltaF508, and G551D mutants. The most active racemates were subjected to chiral separation by HPLC, and the pure enantiomers were tested to evaluate any gains in activity. Although three enantiomers demonstrated high potency (K(d) values less than 0.09, 0.1, and 0.5 muM in G1349D, DeltaF508, and G551D, respectively), in general, the screening of pure enantiomers did not produce a great diversity in potency values. It is probable that the degree of DHP asymmetry considered in our analysis is still insufficient with respect to that allowed in a putative DHP binding site in CFTR, so that the site could equally accommodate both enantiomers.

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6 [4-6] Compounds able to repair class II CFTR mutations (among which the most relevant is DF508) are defined as "correctors", whereas compounds able to ameliorate the gating defect in class III mutations (which include G551D and G1349D) are defined as "potentiators". Login to comment
10 [14] These defects can be corrected by the same potentiators that are effective on G551D and G1349D mutations. Login to comment
16 A small set of asymmetric 4-aryl-DHPs was synthesized, and each racemic couple was tested in a functional assay carried out on cells expressing the G1349D, DF508, and G551D mutants. Login to comment
18 Although three enantiomers demonstrated high potency (Kd values less than 0.09, 0.1, and 0.5 mm in G1349D, DF508, and G551D, respectively), in general, the screening of pure enantiomers did not produce a great diversity in potency values. Login to comment
49 Results Biological results of racemates Compounds 4a-l were first tested on Fischer rat thyroid (FRT) cells expressing the G1349D mutation to evaluate, by means of the iodide influx assay, whether the synthesized DHPs could ameliorate the gating defect of mutant CFTR. Login to comment
65 Evaluation of G1349D CFTR gating effect by compounds 4a-l tested as racemates. Login to comment
78 mers; compounds (À)-4a, (À)-4c, (+)-4d, (+)-4e, and (À)-4g generally show a slightly higher activity than their enantiomeric counterparts in all cell lines. In particular, the Kd ratios of the above values range from 1.7 to 4.7 for G1349D, from 2.0 to 3.1 for rescued DF508, and from 1.5 to 5.5 for G551D. Login to comment
95 Evaluation of G1349D, rescued DF508, and G551D CFTR gating effects by selected DHPs tested as pure enantiomers. Login to comment
96 [a] G1349D Rescued DF508 G551D Compd Kd [mm] Emax [msÀ1 ] Kd [mm] Emax [msÀ1 ] Kd [mm] Emax [msÀ1 ] (+)-4a 0.362Æ0.125 126Æ9.8 1.0Æ0.3 29Æ2.2 1.8Æ0.40 18Æ1.6 (À)-4a 0.210Æ0.085 121Æ8.0 0.5Æ0.2 28Æ1.9 0.6Æ0.20 18Æ1.5 (+)-4c 0.088Æ0.055 123Æ8.3 0.16Æ0.02 28Æ2.3 1.1Æ0.30 18Æ1.1 (À)-4c 0.036Æ0.025 120Æ7.8 0.082Æ0.011 27Æ2.0 0.20Æ0.04 18Æ1.0 (+)-4d 0.046Æ0.021 97Æ6.7 0.061Æ0.022 28Æ2.4 0.46Æ0.10 23Æ1.9 (À)-4d 0.216Æ0.031 109Æ8.1 0.187Æ0.025 25Æ2.1 0.77Æ0.12 19Æ1.6 (+)-4e 0.080Æ0.055 130Æ10.2 0.09Æ0.2 30Æ2.5 0.14Æ0.04 17Æ1.6 (À)-4e 0.158Æ0.045 125Æ11.3 0.25Æ0.03 29Æ2.3 0.24Æ0.08 16Æ1.1 (+)-4g 0.559Æ0.155 121Æ9.4 1.4Æ0.3 28Æ2.0 2.3Æ0.5 19Æ1.2 (À)-4g 0.313Æ0.075 122Æ7.2 0.48Æ0.17 30Æ2.5 1.5Æ0.4 21Æ1.8 [a] Kd and Emax values are the mean ÆSEM of n=5-10 experiments. Login to comment
98 [22-24] In particular, compounds 4c, 4d, and 4e show activity at concentrations <0.1 mm in the G1349D cell line, giving evidence, in this small series, for the importance of a branched substituent at 4`. Login to comment
100 On the other hand, 4-naphthyl derivatives 4 f-i showed moderate activity (only 4g has a Kd value lower than 0.4 mm in the G1349D cell line). Login to comment
102 Compounds that showed the best values in the G1349D cell line (Kd <0.4 mm) were also tested against rescued DF508 and G551D mutations, demonstrating high potency (Table 2). Login to comment
105 Interestingly, we found that the compounds appear to be more active toward G1349D than toward the DF508 mutant. Login to comment
106 This is not surprising, as these two mutations affect different NBDs (NBD1 for DF508 and NBD2 for G1349D); therefore, they may affect the potentiator binding site(s) differently (possibly localized at the NBD1/NBD2 interface). Login to comment
112 The biological tests on FRT cells were repeated for each separated enantiomer, and the results listed in Table 4 indicate a clear, although not dramatic, increase in activity for compounds (À)-4a, (À)-4c, (+)-4d, (+)-4e, and (À)-4g with respect to the enantiomeric counterparts in all cell lines. In particular, compounds (À)-4c and (+)-4d showed Kd values lower than 0.050 and 0.09 mm in G1349D and rescued DF508, respectively, whereas compound (+)-4e had Kd <0.15 mm in G551D. Login to comment
165 Biology CFTR assays Cell culture: Fischer rat thyroid (FRT) cells, stably transfected with G1349D, G551D, or DF508 CFTR and the halide-sensitive yellow fluorescent protein YFP-H148Q/I152L[32] were cultured in Coon`s modified Ham`s F-12 medium supplemented with 10% fetal calf serum, l-glutamine (2 mm), penicillin (100 UmLÀ1 ), and streptomycin (100 mgmLÀ1 ). Login to comment
170 Fluorescence assay for CFTR activity: Measurements of CFTR activity were carried out on FRT cells expressing G1349D, G551D, or DF508 CFTR and the halide-sensitive YFP 48 h after plating on microplates. Login to comment

[hide] Ligand-based design, in silico ADME-Tox filtering,... Eur J Med Chem. 2012 Sep;55:188-94. doi: 10.1016/j.ejmech.2012.07.017. Epub 2012 Jul 23. Visentin S, Ermondi G, Medana C, Pedemonte N, Galietta L, Caron G
Ligand-based design, in silico ADME-Tox filtering, synthesis and biological evaluation to discover new soluble 1,4-DHP-based CFTR activators.
Eur J Med Chem. 2012 Sep;55:188-94. doi: 10.1016/j.ejmech.2012.07.017. Epub 2012 Jul 23., [PMID:22889557]

Abstract [show]
The altered gating of the mutant CFTR chloride channel cystic fibrosis (CF) may be corrected by small molecules called potentiators. We present a molecular scale simulation system for the discovery of DeltaF508-CFTR soluble potentiators. Results report the design, ADME-Tox prediction, synthesis, solubility determination and in vitro biological evaluation of two 1,4-dihydropyridines (DHPs). Compound 1 shows a promising ADME-Tox profile and good potency.

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10 Instead, other mutations such as G551D and G1349D cause only a gating defect. Login to comment

[hide] Ivacaftor potentiation of multiple CFTR channels w... J Cyst Fibros. 2012 May;11(3):237-45. doi: 10.1016/j.jcf.2011.12.005. Epub 2012 Jan 30. Yu H, Burton B, Huang CJ, Worley J, Cao D, Johnson JP Jr, Urrutia A, Joubran J, Seepersaud S, Sussky K, Hoffman BJ, Van Goor F
Ivacaftor potentiation of multiple CFTR channels with gating mutations.
J Cyst Fibros. 2012 May;11(3):237-45. doi: 10.1016/j.jcf.2011.12.005. Epub 2012 Jan 30., [PMID:22293084]

Abstract [show]
BACKGROUND: The investigational CFTR potentiator ivacaftor (VX-770) increased CFTR channel activity and improved lung function in subjects with CF who have the G551D CFTR gating mutation. The aim of this in vitro study was to determine whether ivacaftor potentiates mutant CFTR with gating defects caused by other CFTR gating mutations. METHODS: The effects of ivacaftor on CFTR channel open probability and chloride transport were tested in electrophysiological studies using Fischer rat thyroid (FRT) cells expressing different CFTR gating mutations. RESULTS: Ivacaftor potentiated multiple mutant CFTR forms with defects in CFTR channel gating. These included the G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P and G1349D CFTR gating mutations. CONCLUSION: These in vitro data suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support investigation of the potential clinical benefit of ivacaftor in CF patients who have CFTR gating mutations beyond G551D.

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4 These included the G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P and G1349D CFTR gating mutations. Login to comment
23 Other known CFTR gating mutations include G178R, G551S, G970R, G1244E, S1255P, and G1349D [9-11]. Login to comment
39 These included G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR [4,7,9-11]. Login to comment
46 This analysis showed that, as expected for known CFTR gating mutations (G551D, G178R, G551S, G970R, G1244E, S1255P, and G1349D) [5,9-11], the amount of CFTR delivered to the cell surface was generally similar between CFTR with gating defects and normal CFTR. Login to comment
50 Ivacaftor increased the channel gating of mutant CFTR with defective channel gating The effect of ivacaftor on CFTR channel gating was monitored by quantifying the channel open probability by patch-clamp electrophysiology using membrane patches excised from FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, or G1349D-CFTR. Login to comment
52 Under these conditions, the baseline CFTR channel open probability of G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR was ≤5% of normal CFTR (Fig. 2, B; Table 1). Login to comment
53 For most mutant CFTR forms, the single channel current amplitude, a measure of channel conductance, was similar to normal CFTR (between 77% and 122% of normal CFTR), although a small but statistically significant difference in single channel current amplitude was observed for S1255P-CFTR (Table 1). Login to comment
58 Ivacaftor enhanced chloride transport through mutant CFTR with defective channel gating The impact of the increase in CFTR channel gating by ivacaftor on total chloride transport was assessed in Ussing chamber studies using FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR. Login to comment
61 Under these conditions, the baseline level of chloride transport in FRT cells expressing G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR was b10% of normal CFTR (Fig. 3; Table 2), which was consistent with the low CFTR channel open probability of these mutant CFTR forms (Table 1). Login to comment
71 Patch-clamp studies confirmed that the channel open probability of S549N-, S549R-, and S1251N-CFTR was b5% of normal CFTR, whereas the single channel current amplitude Normal F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 50 100 150 200 CFTRmRNA (%NormalCFTR) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 ** * CFTRMaturation (Mature/Total) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 100 200 300 400 ** * * * CFTR Mutations MatureCFTR (%NormalCFTR) A B D C Mature Immature Fig. 1. Login to comment
90 In a panel of FRT cells expressing G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR, we confirmed that all these mutant CFTR forms shared similar in vitro functional characteristics that were consistent with a defect in channel gating. Login to comment
91 In addition, we showed that the 3 additional mutations, S549N, S549R, and S1251N also have characteristics consistent with gating defects. Login to comment
93 Ivacaftor addition caused a N10-fold increase in CFTR-mediated chloride transport in FRT cells expressing G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR. Login to comment
96 Taken together, these in vitro results provide a rationale for testing the potential benefit of ivacaftor in individuals with CF who have a CFTR gating mutation other than G551D, including the G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P, and G1349D CFTR gating mutations. Login to comment
97 Evaluation of CF-associated CFTR mutations that were expected to cause protein alterations in the ATP-binding sites formed by the NBDs indicated that S549N- and S1251N-CFTR also shared similar in vitro functional characteristics with G551D-CFTR and could be classified as CFTR gating mutations. Login to comment
99 The partial reduction in S549R-CFTR maturation was ~27% of A Normal G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 Baseline With 10 µM Ivacaftor * * * * * * * * * * * CFTR Mutation ChannelOpenProbability ChannelOpenProbability (%NormalCFTR) B 1pA 3sec + 10 µM Ivacaftor G1349D S1255P G970R G551S G178R G1244E Baseline Normal G551D S1251N S549N S549R Fig. 2. Login to comment
122 These included derivatives of 1,4-dihydropyridine and phenylglycine which potentiated G551D-, G970R-, and G1349D-CFTR to a similar extent as ivacaftor [23,25-26]. Login to comment
123 In contrast, sulfamoyl-4-oxoquinoline-3-carboxamides were weakly effective on G551D-, G970R-, and G1349D-CFTR [26] and phloxine B strongly potentiated G551D-CFTR, but not G1349D-CFTR [22]. Login to comment
127 Like G551D, the G551S, G1244E, S1255P, and G1349D CFTR gating mutations, as well as the S549N, S549R, and S1251N CFTR gating mutations identified in the Table 1 Effect of ivacaftor on the channel gating activity of CFTR with gating mutations. Login to comment
128 Single channel current amplitude at 80 mV CFTR channel open probability Baseline With 10 μM ivacaftor Baseline With 10 μM ivacaftor Mutation pA % Normal pA % Normal Po % Normal Po % Normal Normal 0.57±0.03 100 0.63±0.02 111 0.400±0.04 100 0.800±0.04 a 200 G551D 0.46±0.06 81 0.46±0.03 81 0.019±0.01 b 5 0.121±0.035 a 30 G178R 0.59±0.11 103 0.66±0.08 116 0.005±0.001 b 1 0.228±0.022 a 57 S549N 0.55±0.02 97 0.61±0.02 108 0.003±0.010 b 1 0.396±0.119 a 99 S549R 0.45±0.01 b 79 0.55±0.02 a 96 0.004±0.010 b 1 0.143±0.031 a 36 G551S 0.57±0.13 100 0.64±0.02 113 0.010±0.001 b 3 0.337±0.110 a 84 G970R 0.55±0.03 96 0.55±0.03 97 0.001±0.001 b 0 0.245±0.042 a 61 G1244E 0.44±0.11 77 0.54±0.08 94 0.011±0.010 b 3 0.470±0.122 a 118 S1251N 0.54±0.07 95 0.63±0.04 111 0.003±0.010 b 1 0.350±0.03 a 88 S1255P 0.70±0.03 b 122 0.71±0.02 125 0.018±0.016 b 5 0.468±0.168 a 117 G1349D 0.49±0.08 85 0.63±0.06 111 0.019±0.015 b 5 0.315±0.110 a 79 a Significantly different (Pb0.05; paired t-test, n=3-5) compared to baseline levels for each CFTR mutation. Login to comment
130 0 100 200 300 400 -9 -8 -7 -6 -5 -4 G178R G551D G551S 0 S549N S549R Ivacaftor [Log M] 0 100 200 300 400 0 50 100 150 200 -9 -8 -7 -6 -5 -4 G970R G1244E S1255P G1349D 0 S1251N Ivacaftor [Log M] ChlorideTransport (%NormalCFTR) Normal Forskolin G178R G551S G970R G1244E 50 2 1 min S1255P Normal F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 100 200 300 400 0 50 100 150 200 * * * * * * * * * * * * * CFTR Mutation ChlorideTransport(µA/cm2)ChlorideTransport(µA/cm2) ChlorideTransport(A/cm2) ChlorideTransport (%NormalCFTR) B G1349D G551D A F508del C S549N S549R S1251N Baseline Baseline present study, cause protein alterations in the ATP binding pockets formed by the two NBDs required for normal CFTR channel gating (Fig. 4) [2]. Login to comment
131 The G178R and G970R CFTR gating mutations alter the intracellular cytoplasmic loops that are believed to link the ATP-driven conformational changes in the NBDs to the opening of the CFTR channel pore formed by the membrane spanning domains [27]. Login to comment
144 The in vitro data presented here suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support the investigation of ivacaftor in patients with CF who have CFTR gating mutations beyond G551D, including G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D. Login to comment
24 Other known CFTR gating mutations include G178R, G551S, G970R, G1244E, S1255P, and G1349D [9-11]. Login to comment
40 These included G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR [4,7,9-11]. Login to comment
47 This analysis showed that, as expected for known CFTR gating mutations (G551D, G178R, G551S, G970R, G1244E, S1255P, and G1349D) [5,9-11], the amount of CFTR delivered to the cell surface was generally similar between CFTR with gating defects and normal CFTR. Login to comment
51 Ivacaftor increased the channel gating of mutant CFTR with defective channel gating The effect of ivacaftor on CFTR channel gating was monitored by quantifying the channel open probability by patch-clamp electrophysiology using membrane patches excised from FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, or G1349D-CFTR. Login to comment
59 Ivacaftor enhanced chloride transport through mutant CFTR with defective channel gating The impact of the increase in CFTR channel gating by ivacaftor on total chloride transport was assessed in Ussing chamber studies using FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR. Login to comment
62 Under these conditions, the baseline level of chloride transport in FRT cells expressing G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR was b10% of normal CFTR (Fig. 3; Table 2), which was consistent with the low CFTR channel open probability of these mutant CFTR forms (Table 1). Login to comment
72 Patch-clamp studies confirmed that the channel open probability of S549N-, S549R-, and S1251N-CFTR was b5% of normal CFTR, whereas the single channel current amplitude Normal F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 50 100 150 200 CFTR mRNA (% Normal CFTR) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 ** * CFTR Maturation (Mature/Total) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 100 200 300 400 ** * * * CFTR Mutations Mature CFTR (% Normal CFTR) A B D C Mature Immature Fig. 1. Login to comment
94 Ivacaftor addition caused a N10-fold increase in CFTR-mediated chloride transport in FRT cells expressing G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR. Login to comment
100 The partial reduction in S549R-CFTR maturation was ~27% of A Normal G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 Baseline With 10 &#b5;M Ivacaftor * * * * * * * * * * * CFTR Mutation Channel Open Probability Channel Open Probability (% Normal CFTR) B 1pA 3sec + 10 &#b5;M Ivacaftor G1349D S1255P G970R G551S G178R G1244E Baseline Normal G551D S1251N S549N S549R Fig. 2. Login to comment
124 In contrast, sulfamoyl-4-oxoquinoline-3-carboxamides were weakly effective on G551D-, G970R-, and G1349D-CFTR [26] and phloxine B strongly potentiated G551D-CFTR, but not G1349D-CFTR [22]. Login to comment
129 Single channel current amplitude at 80 mV CFTR channel open probability Baseline With 10 bc;M ivacaftor Baseline With 10 bc;M ivacaftor Mutation pA % Normal pA % Normal Po % Normal Po % Normal Normal 0.57&#b1;0.03 100 0.63&#b1;0.02 111 0.400&#b1;0.04 100 0.800&#b1;0.04 a 200 G551D 0.46&#b1;0.06 81 0.46&#b1;0.03 81 0.019&#b1;0.01 b 5 0.121&#b1;0.035 a 30 G178R 0.59&#b1;0.11 103 0.66&#b1;0.08 116 0.005&#b1;0.001 b 1 0.228&#b1;0.022 a 57 S549N 0.55&#b1;0.02 97 0.61&#b1;0.02 108 0.003&#b1;0.010 b 1 0.396&#b1;0.119 a 99 S549R 0.45&#b1;0.01 b 79 0.55&#b1;0.02 a 96 0.004&#b1;0.010 b 1 0.143&#b1;0.031 a 36 G551S 0.57&#b1;0.13 100 0.64&#b1;0.02 113 0.010&#b1;0.001 b 3 0.337&#b1;0.110 a 84 G970R 0.55&#b1;0.03 96 0.55&#b1;0.03 97 0.001&#b1;0.001 b 0 0.245&#b1;0.042 a 61 G1244E 0.44&#b1;0.11 77 0.54&#b1;0.08 94 0.011&#b1;0.010 b 3 0.470&#b1;0.122 a 118 S1251N 0.54&#b1;0.07 95 0.63&#b1;0.04 111 0.003&#b1;0.010 b 1 0.350&#b1;0.03 a 88 S1255P 0.70&#b1;0.03 b 122 0.71&#b1;0.02 125 0.018&#b1;0.016 b 5 0.468&#b1;0.168 a 117 G1349D 0.49&#b1;0.08 85 0.63&#b1;0.06 111 0.019&#b1;0.015 b 5 0.315&#b1;0.110 a 79 a Significantly different (Pb0.05; paired t-test, n=3-5) compared to baseline levels for each CFTR mutation. Login to comment
145 The in vitro data presented here suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support the investigation of ivacaftor in patients with CF who have CFTR gating mutations beyond G551D, including G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D. Login to comment

[hide] Identification of a novel post-hydrolytic state in... J Gen Physiol. 2012 May;139(5):359-70. doi: 10.1085/jgp.201210789. Epub 2012 Apr 16. Jih KY, Sohma Y, Li M, Hwang TC
Identification of a novel post-hydrolytic state in CFTR gating.
J Gen Physiol. 2012 May;139(5):359-70. doi: 10.1085/jgp.201210789. Epub 2012 Apr 16., [PMID:22508846]

Abstract [show]
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC protein family may share an evolutionary origin. Although ABC exporters harness the free energy of ATP hydrolysis to fuel a transport cycle, for CFTR, ATP-induced dimerization of its nucleotide-binding domains (NBDs) and subsequent hydrolysis-triggered dimer separation are proposed to be coupled, respectively, to the opening and closing of the gate in its transmembrane domains. In this study, by using nonhydrolyzable ATP analogues, such as pyrophosphate or adenylyl-imidodiphosphate as baits, we captured a short-lived state (state X), which distinguishes itself from the previously identified long-lived C2 closed state by its fast response to these nonhydrolyzable ligands. As state X is caught during the decay phase of channel closing upon washout of the ligand ATP but before the channel sojourns to the C2 closed state, it likely emerges after the bound ATP in the catalysis-competent site has been hydrolyzed and the hydrolytic products have been released. Thus, this newly identified post-hydrolytic state may share a similar conformation of NBDs as the C2 closed state (i.e., a partially separated NBD and a vacated ATP-binding pocket). The significance of this novel state in understanding the structural basis of CFTR gating is discussed.

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304 Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Login to comment

[hide] Genotype-phenotype correlation in cystic fibrosis ... Genet Mol Biol. 2011 Jul;34(3):416-20. Epub 2011 Jul 1. Polizzi A, Tesse R, Santostasi T, Diana A, Manca A, Logrillo VP, Cazzato MD, Pantaleo MG, Armenio L
Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele.
Genet Mol Biol. 2011 Jul;34(3):416-20. Epub 2011 Jul 1., [PMID:21931512]

Abstract [show]
Cystic fibrosis (CF) is caused by CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations. We ascertained five patients with a novel complex CFTR allele, with two mutations, H939R and H949L, inherited in cis in the same exon of CFTR gene, and one different mutation per patient inherited in trans in a wide population of 289 Caucasian CF subjects from South Italy. The genotype-phenotype relationship in patients bearing this complex allele was investigated. The two associated mutations were related to classical severe CF phenotypes.

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60 The other four patients were compound heterozygotes respectively for G542X, 1259insA, G1349D, F508del and the two associated mutation in exon 15 [H939R;H949L]. Login to comment
61 The mutations G542X, 1259insA, G1349D, F508del have already been described as severe CF-asssociated mutation (Casals et al., 1993; Morral et al., 1993; Morral et al., 1994; Kerem et al., 1995; Estivill et al., 1997; Shrimpton et al., 1997; Rowntree and Harris 2003; Bompadre et al., 2007; Castellani et al., 2008). Login to comment
62 Particularly, 1259insA and G1349D represent with few other mutations, 4382delA, I502T, 852del22, 4016insT, D579G, R1158X and L1077P, almost 20% of the CF alleles found in the Apulian population (Castaldo et al., 2005; Polizzi et al., 2005). Login to comment
65 On the other hand, the F508del mutation, a deletion of three bases encoding a phenylalanine residue at position 508 within the first nucleotide binding domain (NBD), affects CFTR maturation (class II mutations) (Rowntree and Harris, 2003), while the G1349D plays a role in ATP-dependent opening of the chloride channel, resulting in a defective CFTR activation 418 Novel complex allele in CF Table 1 - Clinical features of five unrelated patients with the complex allele [H939R;H949L]. Login to comment
66 Patients characteris* Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Mutation in trans with [H939R;H949L] R248T G542X 1259insA G1349D F508del Sex male male male male male Present age (years) 15 15 17 20 25 Age at diagnosis (years) 14 3 0 10 10 Airways colonization No SA SA SA PA, BC Age of first colonization (years) / 9 6 12 14 BMI (kg/m2 ) 21.9 17.0 15.1 17.6 17.5 FEV1 as % predicted 84.4 114.8 80.9 93.2 53.7 Sweat chloride concentration (mEq/L) 78 100 108 92 95 S-K score 100 70 60 75 40 Brasfield scorez N/A 5 11 7 21 Pancreas status PS PI PI PI PI Diagnosis CFTR-RD CF CF CF CF SA = Staphilococcus aureus, PA = Pseudomonas aeruginosa, BC = Burkholderia cepacia; N/A = not applicable; S-K = Shwachman-Kulczycki: the system is based on four parameters (general activity, physical examination, growth and nutrition and chest radiograph x-ray), and is rated as a) excellent: 86-100 b) good: 71-85, c) mild: 56-70, d) moderate: 41-55, and e) severe: < 40 (Shwachman and Kulczyzki, 1958); z scoring system from 3 "mild" to 25 "most severe" (Brett et al., 1992) after x-ray; PS/PI = Pancreatic sufficiency/insufficiency. Login to comment
71 In our study, the four patients carrying the complex allele [H939R;H949L] associated in trans with the severe mutations G542X, 1259insA, G1349D and F508del presented the classic CF phenotype. Login to comment

[hide] Molecular modeling of the heterodimer of human CFT... J Mol Graph Model. 2009 Apr;27(7):822-8. Epub 2008 Dec 24. Huang SY, Bolser D, Liu HY, Hwang TC, Zou X
Molecular modeling of the heterodimer of human CFTR's nucleotide-binding domains using a protein-protein docking approach.
J Mol Graph Model. 2009 Apr;27(7):822-8. Epub 2008 Dec 24., [PMID:19167254]

Abstract [show]
We have presented a new protein-protein docking approach to model heterodimeric structures based on the conformations of the monomeric units. The conventional modeling method relies on superimposing two monomeric structures onto the crystal structure of a homologous protein dimer. The resulting structure may exhibit severe backbone clashes at the dimeric interface depending on the backbone dissimilarity between the target and template proteins. Our method overcomes the backbone clashing problem and requires no a priori knowledge of the dimeric structure of a homologous protein. Here we used human Cystic Fibrosis Transmembrane conductance Regulator (CFTR), a chloride channel whose dysfunction causes cystic fibrosis, for illustration. The two intracellular nucleotide-binding domains (NBDs) of CFTR control the opening and closing of the channel. Yet, the structure of the CFTR's NBD1-NBD2 complex has not been experimentally determined. Thus, correct modeling of this heterodimeric structure is valuable for understanding CFTR functions and would have potential applications for drug design for cystic fibrosis treatment. Based on the crystal structure of human CFTR's NBD1, we constructed a model of the NBD1-NBD2 complex. The constructed model is consistent with the dimeric mode observed in the crystal structures of other ABC transporters. To verify our structural model, an ATP substrate was docked into the nucleotide-binding site. The predicted binding mode shows consistency with related crystallographic findings and CFTR functional studies. Finally, genistein, an agent that enhances CFTR activity, though the mechanism for such enhancement is unclear, was docked to the model. Our predictions agreed with genistein's bell-shaped dose-response relationship. Potential mutagenesis experiments were proposed for understanding the potentiation mechanism of genistein and for providing insightful information for drug design targeting at CFTR. The method used in this study can be applied to modeling studies of other dimeric protein structures.

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205 The interactions between the Q-loop and signature motif may explain why G551D and G1349D, the CF-associated mutations in the signature motif, influence genistein`s effects [49,13]. Login to comment
201 The interactions between the Q-loop and signature motif may explain why G551D and G1349D, the CF-associated mutations in the signature motif, influence genistein`s effects [49,13]. Login to comment

[hide] Molecular basis for the ATPase activity of CFTR. Arch Biochem Biophys. 2008 Aug 1;476(1):95-100. Epub 2008 Apr 8. Cheung JC, Kim Chiaw P, Pasyk S, Bear CE
Molecular basis for the ATPase activity of CFTR.
Arch Biochem Biophys. 2008 Aug 1;476(1):95-100. Epub 2008 Apr 8., [PMID:18417076]

Abstract [show]
CFTR is a member of the ABC (ATP binding cassette) superfamily of transporters. It is a multidomain membrane protein, which utilizes ATP to regulate the flux of its substrate through the membrane. CFTR is distinct in that it functions as a channel and it possesses a unique regulatory R domain. There has been significant progress in understanding the molecular basis for CFTR activity as an ATPase. The dimeric complex of NBD structures seen in prokaryotic ABC transporters, together with the structure of an isolated CF-NBD1, provide a unifying molecular template to model the structural basis for the ATPase activity of CFTR. The dynamic nature of the interaction between the NBDs and the R domain has been revealed in NMR studies. On the other hand, understanding the mechanisms mediating the transmission of information from the cytosolic domains to the membrane and the channel gate of CFTR remains a central challenge.

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129 This latter concept is supported by the idea that disruption of the signature motifs in each site, G551D (Site A) and G1349D (Site B) led to differential consequences in gating. Login to comment
130 The Site A mutant: G551D completely lacked ATP-dependent opening (and ADP-dependent inhibition) whereas the Site B mutant: G1349D exhibited only partially reduced nucleotide-dependent opening. Login to comment
131 The partial consequences of G1349D on gating suggest a possible role of Site B in modulating the activity of Site A. Login to comment

[hide] Rapid screening for 31 mutations and polymorphisms... Methods Mol Med. 2005;114:147-71. Dunbar SA, Jacobson JW
Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array.
Methods Mol Med. 2005;114:147-71., [PMID:16156102]

Abstract [show]
A suspension array hybridization assay is described for the detection of 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator (CFTR) gene using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for design of oligonucleotide capture probes and PCR amplification primers, coupling oligonucleotide capture probes to carboxylated microspheres, hybridization of coupled microspheres to oligonucleotide targets, production of targets from DNA samples by multiplexed PCR amplification, and detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. Mutation screening with the system is rapid, requires relatively few sample manipulations, and provides adequate resolution to reliably genotype the 25 CFTR mutations and 6 CFTR polymorphisms contained in the ACMG/ACOG/NIH-recommended core mutation panel for general population CF carrier screening.

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118 Table 5 Genomic DNA Samples CFTR genotype Sourcea Normal/normal Sigma, D6537 ΔF508/normal Patient sample ΔF508/ΔF508 Coriell Cell Repositories, NA04540 ΔI507/normal Coriell Cell Repositories, NA11277 W1282/normal Coriell Cell Repositories, NA11723 1717-1G→A/normal Coriell Cell Repositories, NA12444 G542X/G542X Coriell Cell Repositories, NA11496B G542X/normal Coriell Cell Repositories, NA11497B ΔF508/G551D Coriell Cell Repositories, NA11274 ΔF508/R553X Coriell Cell Repositories, NA07469 G551D/R553X Coriell Cell Repositories, NA11761 ΔF508/R560T Coriell Cell Repositories, NA11284 ΔF508/R117H Coriell Cell Repositories, NA13591 I148T/normal Patient sample ΔF508/621+1G→T Coriell Cell Repositories, NA11281 N1303K/G1349D Coriell Cell Repositories, NA11472A ΔF508/1078delT Patient sample R334W/? Login to comment

[hide] Microsphere bead arrays and sequence validation of... J Mol Diagn. 2004 Nov;6(4):348-55. Hadd AG, Laosinchai-Wolf W, Novak CR, Badgett MR, Isgur LA, Goldrick M, Walkerpeach CR
Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms.
J Mol Diagn. 2004 Nov;6(4):348-55., [PMID:15507674]

Abstract [show]
The development of simple and rapid methods for the detection of the common genetic mutations associated with cystic fibrosis (CF) requires access to positive-control samples including the 5/7/9T variants of intron 8. We used PCR and a simple multiplex bead-array assay to identify 5/7/9T control samples from 29 commercially available DNA samples. Unpurified PCR products were directly hybridized to color-coded beads containing allele-specific capture probes for 5/7/9T detection. The performance of the assay was investigated using reverse-complement oligonucleotides, individual PCR products, and multiplex PCR products for 5/7/9T detection within a complex CFTR screening assay. Samples were genotyped by grouping the relative signal intensities from each capture probe. Of 29 commercially available DNA samples analyzed, 2 5T/7T, 2 5T/9T, 9 7T/9T, 11 7T/7T, and 5 9T/9T genotypes were identified. The genotype within each sample group was confirmed by DNA sequencing. The assay was compatible with the analysis of 10 to 1000 ng of genomic DNA isolated from whole blood and allowed for the separate identification of primary CFTR mutations from reflex variants. The correct identification of positive controls demonstrated the utility of a simple bead-array assay and provided accessible samples for assay optimization and for routine quality control in the clinical laboratory.

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197 Intron 8 Genotype by Coriell Number, Characterized CF Mutation and Allele Fraction for 5/7/9T Intron 8 genotype Coriell sample Characterized mutation Allele fraction by probe 5T 7T 9T 7T/7T NA09947 Normal 0.04 0.93 0.03 NA11277 ⌬I507/normal 0.06 0.90 0.04 NA11761 G551D/R553X 0.06 0.92 0.02 NA11859 2789ϩ5GϾA/2789ϩ5GϾA 0.02 0.96 0.02 NA11860 3849ϩ10kbCϾT/3849ϩ10kbCϾT 0.03 0.94 0.03 NA12444 1717-1GϾT/normal 0.06 0.87 0.07 NA12585 R1162X/normal 0.07 0.86 0.08 NA12785 R347P/G551D 0.04 0.92 0.05 NA12960 R334W/normal 0.06 0.92 0.02 NA12961 V520F/normal 0.06 0.89 0.05 NA13033 F508C/normal 0.03 0.93 0.04 9T/9T NA01531 ⌬F508/⌬F508 0.14 0.04 0.82 NA11281 621ϩ1GϾT/⌬F508 0.14 0.04 0.82 NA11283 A455E/⌬F508 0.13 0.05 0.82 NA11290 A455E/621ϩ1GϾT 0.12 0.01 0.87 NA11496 G542X/G542X 0.14 0.05 0.81 5T/7T NA11723 W1282X/normal 0.53 0.44 0.03 NA13032 I506V/normal 0.58 0.39 0.03 5T/9T NA11279 129GϾC/⌬F508 0.51 0.00 0.49 NA13591 R117H/⌬F508 0.52 0.00 0.48 7T/9T NA07441 3120ϩ1GϾA/621ϩ1GϾA 0.08 0.41 0.51 NA07552 R553X/⌬F508 0.09 0.36 0.55 NA07830 556dA/⌬F508 0.11 0.37 0.52 NA11275 3659dC/⌬F508 0.10 0.37 0.53 NA11278 Q493X/⌬F508 0.09 0.38 0.53 NA11280 711ϩ1GϾT/621ϩ1GϾA 0.09 0.37 0.54 NA11282 G85E/621ϩ1GϾA 0.07 0.39 0.53 NA11284 R560T/⌬F508 0.08 0.39 0.52 NA11472 N1303K/G1349D 0.08 0.39 0.54 Figure 3. Login to comment

[hide] Identification of CFTR activators and inhibitors: ... Curr Opin Pharmacol. 2004 Oct;4(5):497-503. Galietta LJ, Moran O
Identification of CFTR activators and inhibitors: chance or design?
Curr Opin Pharmacol. 2004 Oct;4(5):497-503., [PMID:15351355]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in various epithelial cells, and is a pharmacological target for activators and inhibitors. Activators are useful for the pharmacotherapy of cystic fibrosis, specifically for those mutations that affect CFTR protein by reducing its ability to stay in the open state. Conversely, inhibitors are potentially useful to treat secretory diarrhoea caused by enterotoxins, as the CFTR is the main route for Cl(-) flux in the intestine. Recently, a variety of potent modulators of the CFTR Cl(-) channel activity have been identified by high-throughput screening of a large collections of small molecules. The identification of CFTR activators and inhibitors with novel chemical scaffolds might help with the rational design of compounds with improved pharmacological properties.

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31 An example is provided by G551D and G1349D mutations, whose severe gating defect is partially corrected by the same openers - the flavonoids genistein and apigenin - that are effective on the F508del mutant [16-19]. Login to comment

[hide] The cystic fibrosis mutation G1349D within the sig... Biochem Pharmacol. 2004 Jun 15;67(12):2187-96. Melin P, Thoreau V, Norez C, Bilan F, Kitzis A, Becq F
The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein.
Biochem Pharmacol. 2004 Jun 15;67(12):2187-96., [PMID:15163550]

Abstract [show]
Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. The class III CF mutations G551D and G1349D are located within the "signature" sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Similar half-maximal effective concentrations (EC(50)) of MPB-91 (22-36microM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. The isoflavone genistein stimulates wild-type (wt)- and delF508-CFTR channel activity in a non-Michaelis-Menten manner. By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Second, genistein stimulates G551D-CFTR without any inhibition at high concentration. We conclude from these results that whereas G551 in NBD1 is an important molecular site for inhibition of CFTR by genistein, the symmetrical G1349 in NBD2 is also one major site but for the activation of CFTR by genistein. Because both mutations alter specifically the mechanism of CFTR channel activation by genistein, we believe that the signature sequences of CFTR act as molecular switches that upon interaction with genistein turn on and off the channel.

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0 The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein Patricia Melin, Vincent Thoreau, Caroline Norez, Fre´de´ric Bilan, Alain Kitzis, Fre´de´ric Becq* Institut de Physiologie et Biologie Cellulaires CNRS UMR 6187, Universite´ de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France Received 27 October 2003; accepted 5 February 2004 Abstract Cystic fibrosis (CF) is a common lethal genetic disease caused by autosomal recessive mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that belongs to the ATP-Binding Cassette (ABC) family of transporters. Login to comment
1 The class III CF mutations G551D and G1349D are located within the ''signature`` sequence LSGGQ and LSHGH of NBD1 and NBD2, respectively. Login to comment
2 We have constructed by site-directed mutagenesis vectors encoding green fluorescent protein (GFP)-tagged wild-type (wt) CFTR or CFTR containing delF508, G551D, G1349D and G551D/G1349D to study their pharmacology after transient expression in COS-7 cells. Login to comment
3 We show that IBMX and the benzo[c]quinolizinium derivative MPB-91 stimulates the activity of G1349D-, G551D- and G551D/G1349D-CFTR only in the presence of cAMP-promoting agents like forskolin or cpt-cAMP. Login to comment
4 Similar half-maximal effective concentrations (EC50) of MPB-91 (22-36 mM) have been determined for wt-, G551D-, G1349D- and G551D/G1349D-CFTR. Login to comment
6 By contrast, the response of G1349D- and G551D-CFTR to genistein is dramatically altered. Login to comment
7 First, genistein is not able to stimulate G1349D- and G551D/G1349D-CFTR. Login to comment
12 Keywords: CFTR; Signature sequences; Genistein; G1349D; G551D; delF508 1. Login to comment
27 The glycine-to-aspartic acid missense mutations G551D and G1349D are class III mutations located within the signature sequence LSGGQ in NBD1 and LSHGH in NBD2, respectively [13-15]. Login to comment
42 The delF508, G551D and G1349D mutations were created using the sense oligonucleotides 50 -CAT- TAAAGAAAATATCATTGGTGTTTCCTATGATG-30 , 50 -GGAATCACACTGAGTGGAGATCAACGAGCAA- GAATTTCTT-30 and 50 -GGCTGTGTCCTAAGCCAT- GACCACAAGCAGTTGATGTGC-30 , respectively, and the corresponding antisense oligonucletotides. Login to comment
43 Double mutant G551D/G1349D was derived from G551D. Login to comment
49 COS-7 cells were either transfected with empty pEGFP-C1 vector (mock) or with expression vector encoding wild-type GFP-tagged CFTR (wt-CFTR), or mutant forms of GFP-CFTR: delF508, G551D, G1349 or the double mutant G551D/G1349D (named 2GD-CFTR). Login to comment
79 Results To study the pharmacology of CFTR, we have introduced green fluorescent protein (GFP)-tagged CFTR proteins into COS-7 cells, i.e. wt-CFTR and four CFTR mutants i.e. delF508, G551D, G1349D and the double mutant G551D/G1349D (named 2GD). Login to comment
85 GFP-G1349D (lane 5), GFP-G551D/G1349D (lane 6) and GFP-G551D (lane 7) appeared with a similar pattern like the non-mutated form of CFTR (lane 3). Login to comment
90 The two mutated channels G1349D- and 2GD-CFTR, like G551D-CFTR, did not respond to forskolin stimulation (n ¼ 4 for each construct, Fig. 2A), as expected from class III CF mutations [13,19]. Login to comment
91 However, when a cAMP cocktail composed of forskolin, IBMX and cpt-cAMP was used instead of forskolin alone, the chloride channel activity of G1349D and 2GD mutants was stimulated, indicating that both proteins have functional cAMP-dependent chloride channel activity (n ¼ 4, Fig. 2B and C) like G551D-CFTR (data not shown and [19]). Login to comment
95 Only glibenclamide inhibited the chloride channel activity of G1349D- and 2GD-CFTR (Fig. 2B and C). Login to comment
96 Note that the vehicle DMSO has no apparent effect on the iodide efflux (Fig. 2D) A summary of the data obtained with glibenclamide and DIDS is presented Fig. 2E for wt-, G1349D- and 2GD-CFTR stimulated by cAMP cocktail. Login to comment
99 We found that MPB-91 stimulated the iodide efflux with similar EC50 of 29 Æ 1:4, 22 Æ 1:4, 22 Æ 1:3 and 36 Æ 1:1 mM for wt-, G551D-, G1349D and 2GD-CFTR, respectively (n ¼ 4 for each concentration). Login to comment
100 We also found that G1349D and the double mutant 2GD could be stimulated by forskolin and 100 mM IBMX (not shown) as previously reported by others [23,24]. Login to comment
109 Lane 1: non transfected COS-7 cells; lane 2: mock-transfected cells; lane 3: GFP-wt-CFTR, lane 4: GFP-delF508-CFTR; lane 5: GFP-G1349D-CFTR; lane 6: GFP-2GD-CFTR; lane 7: GFP-G551D-CFTR. Login to comment
110 (C) confocal imaging showing plasma membrane localization of wt-CFTR, G551D-, G1349D- and 2GD-CFTR. Login to comment
120 The calculated half-maximal effective concentration EC50 was 13 Æ 1:25 mM (n ¼ 4), in perfect agreement with the value of 11 mM determined in our previous study using CHO cells stably expressing 0 2 4 6 8 0.0 0.1 0.2 0.3 cAMP agents +glibenclamide +DIDS time (min) k(min -1 ) G1349D-CFTR 2GD-CFTR 0 2 4 6 8 0.0 0.1 0.2 0.3 cAMP agents + glibenclamide +DIDS time (min) k(min -1 ) 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 mock Fsk 10µM G1349D G551D 2GD wt-CFTR time (min) k(min -1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 0.4 delF508 2GD G551D G1349D DMSO 1% wt time (min) k(min -1 ) cAMP agents + DIDS + glib 0 50 100 wt-CFTR 2GD G1349D %ofmaximalactivation *** ns (A) (B) (C) (D) (E) Fig. 2. Login to comment
121 Functional expression and pharmacology of GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR. Login to comment
123 B and C, analysis of the expression of G1349D-CFTR (B) and 2GD-CFTR (C) mutants in COS-7 cells. Login to comment
127 (E) summary of the % of maximal activation in the presence of glibenclamide and DIDS for wt-, G1349D and 2GD-CFTR, as indicated. Login to comment
132 On the contrary, analysing the effect of genistein on the channel activity of the LSHGH mutant G1349D and of the double mutant 2GD demonstrated their inability to be stimulated even in the presence of 10 mM forskolin and 300 mM genistein. Login to comment
134 Lack of response given by G1349D-expressing cells was observed in the presence of six different concentrations of genistein (with 10 mM forskolin) and was not statistically different from the control, i.e. with forskolin but without genistein. Login to comment
136 The corresponding concentration-dependent activation relationships for wt, G551D and G1349D are superimposed on the same graph in Fig. 4E. Login to comment
138 Since there is no shift to the right of the curve for G1349D, we concluded that the mutant CFTR was fully refractory to genistein stimulation. Login to comment
139 Finally, similar experiments were conducted with CFTR chloride channels having the class II CF mutation delF508 because this mutation belongs to a different class of mutation but causes, like G551D and G1349D, a severe CF phenotype. Login to comment
145 Inconclusion,wehavestudiedthepharmacologyofCFTR chloride channel and the consequence of the three severe CF mutations, delF508, G551D and G1349D. Login to comment
146 We found a dramatic modification of the pharmacological behaviour of CFTR only with the class III mutations G551D and G1349D that appears to be restricted to genistein. Login to comment
160 Thus, deleting the phenylalanine at position 508, which alters the gating mechanism of the channel and the intracellular trafficking process of the protein, has no apparent effect -6 -5 -4 -3 0.00 0.05 0.10 0.15 0.20 wt-CFTR G551D G1349D 2GD Log [MPB-91] (M) kpeak-kbasal(min -1 ) Fig. 3. Login to comment
161 Effect of the benzoquinolizinium activator MPB-91 on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment
165 Calculated EC50 are 29 Æ 1:4 mM (wt-CFTR), 22 Æ 1:4 mM (G551D-), 22 Æ 1:3 mM (G1349D-), and 36 Æ 1:1 mM (2GD-CFTR). Login to comment
167 Some error bars are smaller than the symbol. 0 2 4 6 8 0.0 0.1 0.2 0.3 +1 µM Gst +10 µM Gst +3 µM Gst +30 µM Gst Fsk 10 µM time (min) k(min -1 ) G551D-CFTR 0 2 4 6 8 0.0 0.1 0.2 0.3 Fsk 500 nM +3 µM Gst +10 µM Gst +30 µM Gst time (min) k(min-1 ) wt-CFTR 0 2 4 6 8 0.0 0.1 0.2 0.3 Fsk 500 nM +30 µM Gst +100 µM Gst +300 µM Gst time (min) k(min-1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 +100 µM Gst +30 µM Gst +300 µM Gst Fsk 10 µM time (min) k(min -1 ) 0 2 4 6 8 0.0 0.1 0.2 0.3 Fsk 10µM + 30 µM Gst time (min) k(min -1 ) 2GD-CFTR 0 2 4 6 8 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 +1 µM Gst +3 µM Gst +10 µM Gst +30 µM Gst +100 µM Gst +300 µM Gst Fsk 10µM time (min) k(min -1 ) G1349D-CFTR -6 -5 -4 -3 0.0 0.1 0.2 0.3 G551D CFTRwt G1349D Log[Gst] (M) kpeak-kbasal(min -1 ) (A) (B) (C) (D) (E) Fig. 4. Login to comment
168 Effect of genistein on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment
169 Iodide efflux curves calculated in response to forskolin and various concentrations of genistein with COS-7 cells transiently expressing GFP-CFTR channels: wt- (A), G551D- (B), G1349D- (C) and 2GD- (D). Login to comment
190 On the contrary, with the mutant G1349D the activation of CFTR was abolished. Login to comment
133 On the contrary, analysing the effect of genistein on the channel activity of the LSHGH mutant G1349D and of the double mutant 2GD demonstrated their inability to be stimulated even in the presence of 10 mM forskolin and 300 mM genistein. Login to comment
135 Lack of response given by G1349D-expressing cells was observed in the presence of six different concentrations of genistein (with 10 mM forskolin) and was not statistically different from the control, i.e. with forskolin but without genistein. Login to comment
137 The corresponding concentration-dependent activation relationships for wt, G551D and G1349D are superimposed on the same graph in Fig. 4E. Login to comment
140 Finally, similar experiments were conducted with CFTR chloride channels having the class II CF mutation delF508 because this mutation belongs to a different class of mutation but causes, like G551D and G1349D, a severe CF phenotype. Login to comment
147 We found a dramatic modification of the pharmacological behaviour of CFTR only with the class III mutations G551D and G1349D that appears to be restricted to genistein. Login to comment
162 Effect of the benzoquinolizinium activator MPB-91 on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment
166 Calculated EC50 are 29 1:4 mM (wt-CFTR), 22 1:4 mM (G551D-), 22 1:3 mM (G1349D-), and 36 1:1 mM (2GD-CFTR). Login to comment
170 Effect of genistein on GFP-tagged wt-, G551D-, G1349D- and 2GD-CFTR chloride channel activity. Login to comment
171 Iodide efflux curves calculated in response to forskolin and various concentrations of genistein with COS-7 cells transiently expressing GFP-CFTR channels: wt- (A), G551D- (B), G1349D- (C) and 2GD- (D). Login to comment
193 On the contrary, with the mutant G1349D the activation of CFTR was abolished. Login to comment

[hide] Cystic fibrosis: a multiple exocrinopathy caused b... Am J Med. 1998 Jun;104(6):576-90. Schwiebert EM, Benos DJ, Fuller CM
Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein.
Am J Med. 1998 Jun;104(6):576-90., [PMID:9674722]

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224 In NBD2, a few key mutations have been found that include missense mutations (G1349D, D1370N, K1250M, K1250Q, G1244E, S1255P) and several nonsense mutations (W1282X, S1255X, W1316X). Login to comment
226 In particular, ⌬F508, G551D, and G1349D have been well studied as severe CF disease-causing mutations. Login to comment
229 An analogous mutation to G551D, G1349D, also occurs in CF patients but in the second NBD. Login to comment
237 Creation of transgenic mice carrying some of the more well-known mutations such as ⌬F508, G551D (93), and G1349D as well as others described below may sort out these issues. Login to comment

[hide] ClC and CFTR chloride channel gating. Annu Rev Physiol. 1998;60:689-717. Foskett JK
ClC and CFTR chloride channel gating.
Annu Rev Physiol. 1998;60:689-717., [PMID:9558482]

Abstract [show]
Chloride channels are widely expressed and play important roles in cell volume regulation, transepithelial transport, intracellular pH regulation, and membrane excitability. Most chloride channels have yet to be identified at a molecular level. The ClC gene family and the cystic fibrosis transmembrane conductance regulator (CFTR) are distinct chloride channels expressed in many cell types, and mutations in their genes are the cause of several diseases including myotonias, cystic fibrosis, and kidney stones. Because of their molecular definition and roles in disease, these channels have been studied intensively over the past several years. The focus of this review is on recent studies that have provided new insights into the mechanisms governing the opening and closing, i.e. gating, of the ClC and CFTR chloride channels.

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289 In CFTR, mutation of G551 or G1349 to aspartic acid (G551D, G1349D; both are CF mutations), decreases ATP binding (149) and the G551D mutation inhibits ATP hydrolysis (151). Login to comment

[hide] Genetic findings in congenital bilateral aplasia o... Hum Mutat. 1998;11(6):480. de Meeus A, Guittard C, Desgeorges M, Carles S, Demaille J, Claustres M
Genetic findings in congenital bilateral aplasia of vas deferens patients and identification of six novel mutatations. Mutations in brief no. 138. Online.
Hum Mutat. 1998;11(6):480., [PMID:10200050]

Abstract [show]
Congential bilateral aplasia of vas deferens (CBAVD), a form of male sterility, has been suggested to represent a "genital" form of cystic fibrosis (CF), as mutations in the CFTR gene have been identified in most patients with this condition. Interestingly, the 5T allele in intron 8 appeared to be the most frequent mutation associated with CBAVD. However, the molecular basis of CBAVD is not completely understood. We have analysed the complete coding and flanking CFTR sequences by PCR-DGGE in 64 men with CBAVD from southern France with the aim to list any sequence alteration. Fourty-two of the 64 patients (65.6%) had mutations on both copies of the CFTR gene, including one patient with two mutations in the same copy (DF508 + A1067T). The 5T allele was present in 21/64 cases (33%). Six of the 28 different mutations identified in this study had never been described previously, and appeared to be specific to CBAVD (P111L, M244K, A1364V, G544V, 2896insAG,-33G->A).

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83 Phenotype CFTRamutations Intron 8, Poly(T) tract 1 3 crisis of acute pancreatitis F508 / L206W 9/7 2 F508 / L206W 9/9 3 frequent bronchitis F508 / R347H 9/9 4 F508 / R347H 9/9 5 F508 / M244K 9/7 6 F508 / A1364V 9/7 7 F508 / D1152H 9/7 8 chronic sinusitis and bronchitis F508 / D1152H 9/7 9 F508 / R117H 9/7 10 F508 / R117H 9/7 11 F508 / M952I 9/7 12 D443Y / G542X 7/9 13 D443Y / G542X 7/9 14 2184delA / D443Y 7/7 15 2184delA / D443Y 7/7 16 R347H / D443Y 9/7 17 seminal vesicles agenesia R117H / G1349D 7/7 18 R117H / G1244E 7/7 19 N1303K / P111L 9/7 20 chronic sinusitis, nasal polyps W1282X / D1152H 7/7 21 chronic sinusitis R347H / Y1092X 7/7 22 seminal vesicles agnesia 297-3C-GTT / 4279insA 7/7 23 G544V / F508C 7/7 24 D1152H / 2896insAG 7-9 25 F508 / - 9/5 26 F508 / - 9/5 27 F508 / - 9/5 28 F508 / - 9/5 29 F508 / - 9/5 30 chronic sinusitis, bronchitis F508 / - 9/5 31 sinusitis and allergy F508 / - 9/5 32 allergy F508 / - 9/5 33 F508 / - 9/5 34 F508 / - 9/5 35 F508 / - 9/5 36 F508 / - 9/5 37 bronchitis, asthma F508 / - 9/5 38 chronic sinusitis F508+A1067T / - 9/5 39 chronic sinusitis D1152H / - 7/5 40 2184delA / - 7/5 41 R764X / - 7/5 42 711+1G-GTT / - 7/5 43 F508 / - 9/7 44 F508 / - 9/7 45 F508 / - 9/7 46 F508 / - 9/9 47 R553X / - 7/7 48 -33G-GTA / - 7/7 49 K710X / - 7/7 50 - / - 5/5 51 - / - 5/7 52 - / - 5/7 53 - / - 7/7 54 - / - 7/7 55 - / - 7/7 56 - / - 7/7 57 - / - 7/7 58 - / - 7/7 59 - / - 7/7 60 - / - 7/7 61 - / - 7/9 62 - / - 7/9 63 NIDDb - / - 7/9 64 - / - 7/9 a : Cystic Fibrosis Transmembrane Regulator gene b : Non Insulino-Dependant Diabetis References Anguiano A, Oates RD, Amos JA, Dean M, Gerrard B, Stewart C, Maher TA, White MB, Milunsky A (1992) Congenital absence of the vas deferens: a primarily genital form of cystic fibrosis. Login to comment

[hide] Purification, characterization, and expression of ... J Bioenerg Biomembr. 1997 Oct;29(5):475-82. Clancy JP, Bebok Z, Sorscher EJ
Purification, characterization, and expression of CFTR nucleotide-binding domains.
J Bioenerg Biomembr. 1997 Oct;29(5):475-82., [PMID:9511932]

Abstract [show]
The nucleotide binding domains (NBDs) within CFTR were initially predicted to lie in the cell cytoplasm, and to gate anion permeability through a pore that was present in membrane spanning alpha helices of the overall polypeptide. Our studies designed to characterize CFTR suggest several important features of the isolated nucleotide binding domain. NBD-1 appears to bind nucleotides with similar affinity to the full-length CFTR protein. In solution, the domain contains a high beta sheet content and self-associates into ordered polymers with molecular mass greater than 300,000 Daltons. The domain is very lipophilic, disrupts liposomes, and readily enters the planar lipid bilayer. Clinically important mutations in the domain may disrupt the nucleotide binding capabilities of the protein, either through a direct effect on the nucleotide binding site, or through effects that influence the overall folding of the domain in vitro. Finally, after expression in human epithelial cells (including epithelial cells from a CF patient), the first nucleotide binding domain targets the plasma membrane even in the absence of other constituents of full-length CFTR and mediates anion permeability in these cells.

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64 To confirm that glycine within this motif was important in nucleotide interactions, the corresponding glycine-aspartic acid replacement was made in CFTR NBD-2 (G1349D), and nucleotide binding was compared with thepurified wild type region. Login to comment
65 The clinically important NBD-2 mutation G1349D led to decreased nucleotide binding by NBD-2. Login to comment
75 NBD-2 polypeptides with and without the G1349D mutation were compared by trinitrophenol ATP binding. Login to comment
76 The glycine-to-aspartic acid mutation at position 1349 substantially decreased the affinity of the second nucleotide binding domain for TNP-ATP. Login to comment

[hide] Distinct spectrum of CFTR gene mutations in congen... Hum Genet. 1997 Sep;100(3-4):365-77. Dork T, Dworniczak B, Aulehla-Scholz C, Wieczorek D, Bohm I, Mayerova A, Seydewitz HH, Nieschlag E, Meschede D, Horst J, Pander HJ, Sperling H, Ratjen F, Passarge E, Schmidtke J, Stuhrmann M
Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.
Hum Genet. 1997 Sep;100(3-4):365-77., [PMID:9272157]

Abstract [show]
Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%-2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the "5T" allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for delta F508 and none was compound heterozygous for delta F508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for delta F508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the "5T allele" was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G-->A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.

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100 Our finding of a German CBAVD patient heterozygous for ∆F508 and K1351E adds evidence to previous indications that ABC signature mutations have less severe consequences in the second than in the first CFTR nucleotide-binding fold (e.g. the CF mutation pair G551D/ G1349D). Login to comment

[hide] Effect of cystic fibrosis-associated mutations in ... J Biol Chem. 1996 Aug 30;271(35):21279-84. Cotten JF, Ostedgaard LS, Carson MR, Welsh MJ
Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 1996 Aug 30;271(35):21279-84., [PMID:8702904]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) contains multiple membrane spanning sequences that form a Cl- channel pore and cytosolic domains that control the opening and closing of the channel. The fourth intracellular loop (ICL4), which connects the tenth and eleventh transmembrane spans, has a primary sequence that is highly conserved across species, is the site of a preserved sequence motif in the ABC transporter family, and contains a relatively large number of missense mutations associated with cystic fibrosis (CF). To investigate the role of ICL4 in CFTR function and to learn how CF mutations in this region disrupt function, we studied several CF-associated ICL4 mutants. We found that most ICL4 mutants disrupted the biosynthetic processing of CFTR, although not as severely as the most common DeltaF508 mutation. The mutations had no discernible effect on the channel's pore properties; but some altered gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate. These effects on activity were similar to those observed with mutations in the nucleotide-binding domains, suggesting that ICL4 might help couple activity of the nucleotide-binding domains to gating of the Cl- channel pore. The data also explain how these mutations cause a loss of CFTR function and suggest that some patients with mutations in ICL4 may have a milder clinical phenotype because they retain partial activity of CFTR at the cell membrane.

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139 This pattern of response for A1067T is similar to what we have found with the NBD mutants G551D, G1244E, and G1349D (8). Login to comment
163 and G1349D, decreased the relative response to PPi. Login to comment
189 The fact that ICL4 and NBD2 mutants reduced the response to PPi and the finding that G1349D and A1067T altered the effect of increasing concentrations of ATP in a similar way further suggest some interaction between ICL4 and the NBDs, particularly NBD2. Login to comment
200 Data are mean Ϯ S.E. of (n) measurements for: wild-type (9), F1052V (3), R1066L (4), A1067T (4), G551S (6), K464A (4), G1349D (5), K1250 M at 5 mM PPi (5), wild-type at 5 mM PPi (16). Login to comment
138 This pattern of response for A1067T is similar to what we have found with the NBD mutants G551D, G1244E, and G1349D (8). Login to comment
162 and G1349D, decreased the relative response to PPi. Login to comment
188 The fact that ICL4 and NBD2 mutants reduced the response to PPi and the finding that G1349D and A1067T altered the effect of increasing concentrations of ATP in a similar way further suggest some interaction between ICL4 and the NBDs, particularly NBD2. Login to comment
199 Data are mean 6 S.E. of (n) measurements for: wild-type (9), F1052V (3), R1066L (4), A1067T (4), G551S (6), K464A (4), G1349D (5), K1250 M at 5 mM PPi (5), wild-type at 5 mM PPi (16). Login to comment

[hide] Disease-associated mutations in the fourth cytopla... J Biol Chem. 1996 Jun 21;271(25):15139-45. Seibert FS, Linsdell P, Loo TW, Hanrahan JW, Clarke DM, Riordan JR
Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity.
J Biol Chem. 1996 Jun 21;271(25):15139-45., [PMID:8662892]

Abstract [show]
A cluster of 18 point mutations in exon 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been detected in patients with cystic fibrosis. These mutations cause single amino acid substitutions in the most C-terminal cytoplasmic loop (CL4, residues 1035-1102) of the CFTR chloride channel. Heterologous expression of the mutants showed that 12 produced only core-glycosylated CFTR, which was retained in the endoplasmic reticulum; the other six mutants matured and reached the cell surface. In some cases substitution of one member of pairs of adjacent residues resulted in misprocessing, whereas the other did not. Thus, the secondary structure of CL4 may contribute crucially to the proper folding of the entire CFTR molecule. Cyclic AMP-stimulated iodide efflux was not detected from cells expressing the misprocessed variants but was from the other six, indicating that their mutations cause relatively subtle channel defects. Consistent with this, these latter mutations generally are present in patients who are pancreatic-sufficient, while the processing mutants are mostly from patients who are pancreatic-insufficient. Single-channel patch-clamp analysis demonstrated that the processed mutants had the same ohmic conductance as wild-type CFTR, but a lower open probability, generally due to an increase in channel mean closed time and a reduction in mean open time. This suggests that mutations in CL4 do not affect pore properties of CFTR, but disrupt the mechanism of channel gating.

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88 Other disease-causing CFTR mutants, which are appropriately processed and trafficked to the plasma membrane, show defective ion conduction properties (e.g. R334W, R347H, and R347P; Sheppard et al., 1993; Tabcharani et al., 1993) or defective regulation of channel activity (e.g. G551S, G1244E, S1255P, and G1349D; Anderson and Welsh, 1992). Login to comment
95 Other disease-causing CFTR mutants, which are appropriately processed and trafficked to the plasma membrane, show defective ion conduction properties (e.g. R334W, R347H, and R347P; Sheppard et al., 1993; Tabcharani et al., 1993) or defective regulation of channel activity (e.g. G551S, G1244E, S1255P, and G1349D; Anderson and Welsh, 1992). Login to comment

[hide] CFTR: the nucleotide binding folds regulate the ac... J Gen Physiol. 1996 Jan;107(1):103-19. Wilkinson DJ, Mansoura MK, Watson PY, Smit LS, Collins FS, Dawson DC
CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.
J Gen Physiol. 1996 Jan;107(1):103-19., [PMID:8741733]

Abstract [show]
The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.

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83 rithmic dose-response plots, but for comparison with rates of activation we sought a more unbiased estimate of Ka that took into account three factors: (1) the activation produced by forskolin alone, (2) the block of CFTR by high concentrations of IBMX, and (3) the fact that for insensitive mutants such as G551D, D572N, and G1349D the dose-response showed no tendency toward saturation at the highest concentrations of IBMX. Login to comment
150 The values listed in Table I show that NBF mutations generally reduced the value of (ko,, + ko~), in some cases by more TABLE I Summary ofActivation and DeactivationDatafor Wild-typeCFFR and Mutants of theInvariant Glycinein NBFI ((;,551)orNBF2 (G1349) CFTR Activation Deactivation klon KA (k,,n+ k,,n) (10 ~miu-1 k,,n kos latency *k,,t~ (raM) n (10-~min l) raM-l) (10-3min 1) (10 ~rain-j) n (min) (10-s min-I) wt 0.65 + 0.08 26 664 _+51 118 _+9 588 +-45 76 + 6 20 6.0 _+0.3 88 -+6 16 G551A 3.0 -+0.5*r 6 104 _+5"r 13 _+0.6*r 65 + 3*z 39 -+2* 5 7.7 +_0.5: 70 -+13: 4 G551S 4.7 +-0.5* 5 82 _+6*r 8 -+0.6*: 42 -+3*: 40 -+3*r 10 3.9 +_0.3*** 88 +-6: 6 G551D 9.3 -+0.01" 6 57 _+9*r 4 -+0.6*: 20 -+3*: 37 -+6"r 5 1.8 _+0.2"~ 84 -+10~ 6 G1349A 1.1 + 0.07*: 5 210 _+24"~ 35 -+4*: 172 -+20*: 38 +-4* 4 1.7 _+0.3"~ 184 + 20*: 5 G1349S 3.5 +-0.3* 4 199 _+46*: 23 -+5*: 117 -+27*r 82 -+19+ 6 2.3 _+0.5*+ 144 -+15": 6 G1349D 9.3 + 0.01" 8 114 _+16*++ 8 -+1": 40 +-6*r 74 -+11~ 5 0.6 -+0.1*++ 286 -+37*: 4 Valuesweredetermined as describedin Methods.The symbols(*) and (~) indicatesignificantdifferencesfrom wild-typeCFFRand the analogousmu- tant, respectively(P< 0.05). Login to comment
178 Substitutions to serine (G1349S) and aspartic acid (G1349D) produced progressive reductions such that the relaxation rate for the least conservative mutation, G1349D, was about twice that for the comparable mutation in NBF1. Login to comment
194 ""'"~"NBF1 NBF2 ,~j:~ ,pit'-" 9 G551S o G1349S 20 jl~ 9 G551 D o G1349D o i, ,,,i,0, ,i,,,,i,,, ,i,, ,,i , ~ ,, B o lO 20 30 40 50 60 ,~ 100 80 E 60 or 40 2o ~ 0 c I0o- 80 " 6o - 40 .z- 20 - o- ictOOO'~ .D-O*'Q / ;~ / Eof 9 . Login to comment
219 This destabilization of the activated state is clearly evident in the representative time courses for deactivation of the mutants G1349S and G1349D (Fig. 5 A). Login to comment
221 The most conservative substitution (G551A) did not appreciably alter the latency. Login to comment
222 The less conservative substitutions (G551S, G551D) progressively decreased the latency, but the reductions were always less than those induced by the corresponding mutations (G1349S, G1349D) in NBF2. Login to comment
236 2O 0 ao•-•lo o - .~ 8o- 0 40-- 2o- =o _: 0-- C lOO- 8o --: 6o --: 40 - 2o - o--: NBF1 NBF2 9 Qs ls o a s49s i ~'~'-,,"".~ 9 G551D = G1349D ~. Login to comment
340 Role of the Invariant Glycine The functional importance of the invariant glycine in NBF1 (G551) or NBF2 (G1349) is evident from the existence of mutations (G551S, G551D, and G1349D) that are associated with cystic fibrosis in humans (Cutting et al., 1990; Kerem et al., 1990; Strong et al., 1991). Login to comment
345 Support for the latter view is provided by the observation that the mutations G551D and G1349D impair ATP binding in isolated fusion proteins of NBF1 and NBF2, respectively (Logan et al., 1994). Login to comment
346 On the other hand, the results of Anderson and Welsh (1992) suggested that the mutations G551S and G1349D reduced the open probability of CFTR C1- channels without changing the K~/2 for the effect of ATP concentration on open probability. Login to comment
362 In addition, concentrations of IBMX above 1 mM are required to achieve significant activation of CFTR mutants such as G551D, D572N, and G1349D (cf. Smit et al., 1993). Login to comment
406 The conserved glycines in NBF1 (G551) and NBF2 (G1349) are both sites of mutations that cause either mild (G551S) or severe (G551D, G1349D) cystic fibrosis (Smitet al., 1993) but have not been associated with protein processing defects such as those that characterize the AF508 mutation. Login to comment
84 rithmic dose-response plots, but for comparison with rates of activation we sought a more unbiased estimate of Ka that took into account three factors: (1) the activation produced by forskolin alone, (2) the block of CFTR by high concentrations of IBMX, and (3) the fact that for insensitive mutants such as G551D, D572N, and G1349D the dose-response showed no tendency toward saturation at the highest concentrations of IBMX. Login to comment
152 The values listed in Table I show that NBF mutations generally reduced the value of (ko,, + ko~), in some cases by more TABLE I Summary ofActivation and DeactivationDatafor Wild-typeCFFR and Mutants of theInvariant Glycinein NBFI ((;,551)orNBF2 (G1349) CFTR Activation Deactivation klon KA (k,,n+ k,,n) (10 ~miu-1 k,,n kos latency *k,,t~ (raM) n (10-~min l) raM-l) (10-3min 1) (10 ~rain-j) n (min) (10-s min-I) wt 0.65 + 0.08 26 664 _+51 118 _+9 588 +-45 76 + 6 20 6.0 _+0.3 88 -+6 16 G551A 3.0 -+0.5*r 6 104 _+5"r 13 _+0.6*r 65 + 3*z 39 -+2* 5 7.7 +_0.5: 70 -+13: 4 G551S 4.7 +-0.5* 5 82 _+6*r 8 -+0.6*: 42 -+3*: 40 -+3*r 10 3.9 +_0.3*** 88 +-6: 6 G551D 9.3 -+0.01" 6 57 _+9*r 4 -+0.6*: 20 -+3*: 37 -+6"r 5 1.8 _+0.2"~ 84 -+10~ 6 G1349A 1.1 + 0.07*: 5 210 _+24"~ 35 -+4*: 172 -+20*: 38 +-4* 4 1.7 _+0.3"~ 184 + 20*: 5 G1349S 3.5 +-0.3* 4 199 _+46*: 23 -+5*: 117 -+27*r 82 -+19+ 6 2.3 _+0.5*+ 144 -+15": 6 G1349D 9.3 + 0.01" 8 114 _+16* + + 8 -+1": 40 +-6*r 74 -+11~ 5 0.6 -+0.1* + + 286 -+37*: 4 Valuesweredetermined as describedin Methods.The symbols(*) and (~) indicatesignificantdifferencesfrom wild-typeCFFRand the analogousmu- tant, respectively(P< 0.05). Login to comment
180 Substitutions to serine (G1349S) and aspartic acid (G1349D) produced progressive reductions such that the relaxation rate for the least conservative mutation, G1349D, was about twice that for the comparable mutation in NBF1. Login to comment
197 ""'"~" NBF1 NBF2 ,~j:~ ,pit'-" 9 G551S o G1349S 20 jl~ 9 G551 D o G1349D o i, ,,,i,0, ,i,,,,i,,, ,i,, ,,i , ~ ,, B o lO 20 30 40 50 60 ,~ 100 80 E 60 o r 40 2o ~ 0 c I0o- 80 " 6o - 40 .z20 - o- ictOOO'~ .D-O*'Q / ;~ / Eof 9 . Login to comment
224 The less conservative substitutions (G551S, G551D) progressively decreased the latency, but the reductions were always less than those induced by the corresponding mutations (G1349S, G1349D) in NBF2. Login to comment
238 2O 0 aoߦ-ߦl o o - .~ 8o- 0 40-- 2o- =o _: 0-- C lOO- 8o --: 6o --: 40 - 2o - o--: NBF1 NBF2 9 Qs ls o a s49s i ~'~'-,,"".~ 9 G551D = G1349D ~. Login to comment
342 Role of the Invariant Glycine The functional importance of the invariant glycine in NBF1 (G551) or NBF2 (G1349) is evident from the existence of mutations (G551S, G551D, and G1349D) that are associated with cystic fibrosis in humans (Cutting et al., 1990; Kerem et al., 1990; Strong et al., 1991). Login to comment
347 Support for the latter view is provided by the observation that the mutations G551D and G1349D impair ATP binding in isolated fusion proteins of NBF1 and NBF2, respectively (Logan et al., 1994). Login to comment
348 On the other hand, the results of Anderson and Welsh (1992) suggested that the mutations G551S and G1349D reduced the open probability of CFTR C1-channels without changing the K~/2 for the effect of ATP concentration on open probability. Login to comment
364 In addition, concentrations of IBMX above 1 mM are required to achieve significant activation of CFTR mutants such as G551D, D572N, and G1349D (cf. Smit et al., 1993). Login to comment
408 The conserved glycines in NBF1 (G551) and NBF2 (G1349) are both sites of mutations that cause either mild (G551S) or severe (G551D, G1349D) cystic fibrosis (Smitet al., 1993) but have not been associated with protein processing defects such as those that characterize the AF508 mutation. Login to comment

[hide] Epitope tagging permits cell surface detection of ... Am J Physiol. 1995 Dec;269(6 Pt 1):C1565-76. Howard M, DuVall MD, Devor DC, Dong JY, Henze K, Frizzell RA
Epitope tagging permits cell surface detection of functional CFTR.
Am J Physiol. 1995 Dec;269(6 Pt 1):C1565-76., [PMID:8572187]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.

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244 We produced four tagged dgCFTR mutants that are associated with disease: AF508, N1303K, G551D, and G1349D, bearing the M2 epitope at position 901. Login to comment
255 Cell surface expression of M2-901/CFTR has been observed in a expression of M2-901-labeled G551D, G1349D, K1250M, and D1370N dgCFTRs correlates with carbohydrate variety of cell types including HEp-2, BSC40, and addition (Fig &A-II). Login to comment
265 A: G551D; B: G1349D; C: K1250M; D: D1370N; E: AF508; F: N1303K. Login to comment

[hide] Search for mutations in pancreatic sufficient cyst... Hum Genet. 1995 Sep;96(3):312-8. Brancolini V, Cremonesi L, Belloni E, Pappalardo E, Bordoni R, Seia M, Russo S, Padoan R, Giunta A, Ferrari M
Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations.
Hum Genet. 1995 Sep;96(3):312-8., [PMID:7544319]

Abstract [show]
A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.

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42 The remaining 19 included R352Q (Cremonesi et al. 1992) (three chromosomes), G85E (Zielenski et al. 1991a), Dl152H (High- Fig. 1 A-C Direct sequencing of PCR products from three cystic fibrosis patients (CF) carrying the W57G (A), E193K (B) and D579G (C) mutations, in parallel with control samples (C) displaying normal sequences (N/N) smith et al., personal communication to the CF Genetic Analysis Consortium), R1066H (Ferec et al. 1992), T338I (Saba et al. 1993), 711 +5G--+A (Gasparini et al., personal communication to the CF Genetic Analysis Consortium), M1V (Cheadle et al. 1993), R334W (Gasparini et al. 1991) (two chromosomes each), 4382delA (Claustres et al. 1993), R1158X (Ronchetto et al. 1992), F1052V (Mercier et al. 1993), G1349D (Beaudet et al. 1991), 1898+3A-+G (Cremonesi et al. 1992), $549N (Cutting et al. 1990), 711+ 3A-->G (Petreska et al. 1994), R347P (Dean et al. 1990), 2789+5G--+A (Highsmith et al. 1990), R1066C (Fanen et al. 1992) and S1251N (K~ilin et al. 1992) (one chromosome each). Login to comment
70 (UN yet unidentified mutation) Patient Genotype after Genotype at the end number preliminary screening of the analysis UN/UN M1V/4382delA 1717-1G---~A/UN 1717-1G---~A/R1066H AF508/UN AF508/D579G UN/UN M1V/UN AF508/UN AF508/UN UN/UN T338I/R1158X UN/UN G85E/71 I+5G---~A UN/UN D1152H/UN AF508/UN AF508/UN AF508/UN AF508/3849+ 10kbC---~T UN/UN 711+3A---~G/UN AF508/UN AF508/F1052V UN/UN R352Q/W57G UN/UN 1898+3A----~G/UN AF508/UN AF508/711+5G--~A G542X/UN G542X/DI 152H AF508/UN AF508/E193K 1717-1G---~A/UN 1717-1G---~A/2789+5A---)G AF508/UN AF508/G1349D AF508/UN AF508/G85E AF508/UN AF508/R347P AF508/UN AF508/R352Q AF508/UN AF508/R352Q AF508/UN AF508/S549N G542X/UN G542X/R1066H AF508/UN AF508/T338I AF508/UN AF508/R334W AF508/UN AF508/R334W AF508/UN AF508/S1251N AF508/UN AF508/R1066C AF508/UN AF508/D579G results) while the remaining three haplotypes had been found in association with other rare mutations, which were excluded by DGGE analysis in these patients (Table 3). Login to comment
85 In total, among the mutations detected in our PS patients, 17 (D579G, E193K, F1052V, 711+5G---~A, G1349D, G85E, R347R R352Q, $549N, 2789+5A---~G, D1152H, R1066H, R334W, T338I, 3849+10kbC---~T, S1251N, R1066C) have been detected in compound heterozygosity with a mutation already classified as severe (AF508, 1717-1G--~A, G542X) and thus can be considered as presumably mild. Login to comment
87 R1066C R1066H F1052V 2789+5A->G D1152H 14a14b 15 1617a 17b 18 19 S1251N ItKbC->T G1349D m III! Login to comment
88 20 21 22 23 24 MEMBRANE SPANNING ATP R DOMAIN MEMBRANE SPANNING ATP BINDING BINDING tide binding folds ($549N and D579G in the NBF I, G1349D and S1251N in the NBF II) and one in intron 19 (3849+10kbC--~T), further confirming that the milder defects mostly affect the membrane spanning domains with a greater incidence on the I TM (Fig. 2). Login to comment

[hide] Mutations in the cystic fibrosis gene in patients ... N Engl J Med. 1995 Jun 1;332(22):1475-80. Chillon M, Casals T, Mercier B, Bassas L, Lissens W, Silber S, Romey MC, Ruiz-Romero J, Verlingue C, Claustres M, et al.
Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens.
N Engl J Med. 1995 Jun 1;332(22):1475-80., [PMID:7739684]

Abstract [show]
BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. METHODS: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. RESULTS: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. CONCLUSIONS: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD: The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men.

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74 OF PATIENTS POLYT GENOTYPE† ⌬F508/R668C ⌬F508/D1152H ⌬F508/D1270N ⌬F508/R75L ⌬F508/R117H ⌬F508/L206W ⌬F508/R258G ⌬F508/S1235R ⌬F508/R347H ⌬F508/R347H R117H/G1349D R117H/712-1G→T G149R/R668C R347H/R1066H R553X/R668C R1070W/2869insG ⌬F508/- G542X/- W1282X/- R334W/- K1060T/- R1162X/- N1303K/- A800G/- ⌬F508/- ⌬F508/- ⌬F508/- ⌬E115/- R117H/- R347H/- G542X/- R553X/- 1677delTA/- 2184delA/- 2789ϩ5G→Α/- S1235R/- W1282X/- -/- -/- -/- -/- 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 22 4 3 1 1 1 1 1 7 1 1 1 1 2 1 1 1 1 1 1 1 3 3 1 19 9T/7T 9T/7T 9T/7T 9T/7T 9T/7T 9T/9T 9T/7T 9T/7T 9T/7T 9T/9T 7T/7T 7T/9T 9T/7T 9T/7T 7T/7T 7T/7T 9T/5T 9T/5T 7T/5T 7T/5T 7T/5T 7T/5T 9T/5T 5T/5T 9T/7T 9T/9T 7T/7T 7T/7T 7T/7T 9T/7T 9T/7T 7T/7T 7T/7T 7T/7T 7T/7T 7T/9T 7T/7T 9T/5T 7T/5T 5T/5T 7T/7T -/- 3 7T/9T *Data were obtained from the Spanish population analyzed in this study. Login to comment

[hide] Mechanism of dysfunction of two nucleotide binding... EMBO J. 1995 Mar 1;14(5):876-83. Sheppard DN, Ostedgaard LS, Winter MC, Welsh MJ
Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency.
EMBO J. 1995 Mar 1;14(5):876-83., [PMID:7534226]

Abstract [show]
Variability in the severity of cystic fibrosis (CF) is in part due to specific mutations in the CF transmembrane conductance regulator (CFTR) gene. To understand better how mutations in CFTR disrupt Cl- channel function and to learn about the relationship between genotype and phenotype, we studied two CF mutants, A455E and P574H, that are associated with pancreatic sufficiency. A455E and P574H are located close to conserved ATP binding motifs in CFTR. Both mutants generated cAMP-stimulated apical membrane Cl- currents in heterologous epithelial cells, but current magnitudes were reduced compared with wild-type. Patch-clamp analysis revealed that both mutants had normal conductive properties and regulation by phosphorylation and nucleotides. These mutants had normal or increased Cl- channel activity: A455E had an open-state probability (Po) similar to wild-type, and P574H had an increased Po because bursts of activity were prolonged. However, both mutants produced less mature glycosylated protein, although levels were greater than observed with the delta F508 mutant. These changes in channel activity and processing provide a quantitative explanation for the reduced apical Cl- current. These data also dissociate structural requirements for channel function from features that determine processing. Finally, the results suggest that the residual function associated with these two mutants is sufficient to confer a milder clinical phenotype and infer approaches to developing treatments.

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126 For example, G551S, G1244E, S1255P and G1349D had a markedly reduced PO at all concentrations of MgATP tested, and S1255P was less potently stimulated by MgATP (Anderson and Welsh, 1992; Smit et al., 1993). Login to comment
127 Therefore, we tested the hypothesis that ATP-dependent regulation of A455E and P574H was altered. Login to comment

[hide] Cystic fibrosis: genotypic and phenotypic variatio... Annu Rev Genet. 1995;29:777-807. Zielenski J, Tsui LC
Cystic fibrosis: genotypic and phenotypic variations.
Annu Rev Genet. 1995;29:777-807., [PMID:8825494]

Abstract [show]
Cystic fibrosis (CF) is a common genetic disorder in the Caucasian population. The gene was identified in 1989 on the basis of its map location on chromosome 7. The encoded gene product, named cystic fibrosis transmembrane conductance regulator (CFTR), corresponds to a cAMP-regulated chloride channel found almost exclusively in the secretory epithelial cells. Although the major mutation that results in a single amino acid deletion (F508) accounts for 70% of the disease alleles, more than 550 additional mutant alleles of different forms have been detected. Many of these mutations can be divided into five general classes in terms of their demonstrated or presumed molecular consequences. In addition, a good correlation has been found between CFTR genotype and one of the clinical variables--pancreatic function status. An unexpected finding, however, is the documentation of CFTR mutations in patients with atypical CF disease presentations, including congenital absence of vas deferens and several pulmonary diseases. Thus, the implication of CFTR mutation is more profound than CF alone.

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631 The range of effects of dysregulation of the channel includes those with a severe lack of function (such as that for G551D), reduced response to ATP stimulation (S1255P), and slight reduction of absolute activity (G551S, G1244E, and G1349D) (7, 63). Login to comment

[hide] Functional roles of the nucleotide-binding folds i... Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7. Smit LS, Wilkinson DJ, Mansoura MK, Collins FS, Dawson DC
Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9963-7., [PMID:7694298]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the traffic ATPase superfamily, possesses two putative nucleotide-binding folds (NBFs). The NBFs are sufficiently similar that sequence alignment of highly conserved regions can be used to identify analogous residues in the two domains. To determine whether this structural homology is paralleled in function, we compared the activation of chloride conductance by forskolin and 3-isobutyl-1-methylxanthine in Xenopus oocytes expressing CFTRs bearing mutations in NBF1 or NBF2. Mutation of a conserved glycine in the putative linker domain in either NBF produced virtually identical changes in the sensitivity of chloride conductance to activating conditions, and mutation of this site in both NBFs produced additive effects, suggesting that in the two NBFs this region plays a similar and critical role in the activation process. In contrast, amino acid substitutions in the Walker A and B motifs, thought to form an integral part of the nucleotide-binding pockets, produced strikingly different effects in NBF1 and NBF2. Substitutions for the conserved lysine (Walker A) or aspartate (Walker B) in NBF1 resulted in a marked decrease in sensitivity to activation, whereas the same changes in NBF2 produced an increase in sensitivity. These results are consistent with a model for the activation of CFTR in which both NBF1 and NBF2 are required for normal function but in which either the nature or the exact consequences of nucleotide binding differ for the two domains.

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68 G551D, associated with severe CF (35, 36), and G1349D, also a CF mutation (37), both exhibited a dramatic reduction in sensitivity (K1l2 = 2.5 0 0 wt (12) 100 E .E CO) NBF1 A A G551A c O G551S V v G551 D NBF2 (8) A-A G1349A (9) * * G1349S (6) '-V G1349D (4) (6) (8) 0.2 0.5 1 IBMX, mM FIG. 2. Login to comment
77 To explore further the relative contributions of the two domains, we measured the activation of Cl- currents in oocytes expressing the double mutants G551S/G1349S and G551D/G1349D. Login to comment
104 Even in the case of a mutation that severely compromised activation, e.g. G1349D, current-voltage plots were indistinguishable from wild type. Login to comment
107 Both glycines were replaced by serines (G551S + G1349S) or aspartates (G551D + G1349D), and the dose-response relationships were constructed as in Fig. 2. Login to comment
119 Anderson and Welsh (28) found that in detached patches from transfected cells, mutation of the conserved glycine (G551S or G1349D) dramatically reduced the value ofthe open probability (PO) in the presence ofPKA and MgATP. Login to comment

[hide] Molecular mechanisms of CFTR chloride channel dysf... Cell. 1993 Jul 2;73(7):1251-4. Welsh MJ, Smith AE
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell. 1993 Jul 2;73(7):1251-4., [PMID:7686820]

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17 Classes of CFTR Mutations That Cause CF Class Defect Examples Do- Fre- Clin- main quency ical Protein production Nonsense mutations Frameshift Splice Processing Conduction 6542X NBDI 3.4 3905 insT NBD2 2.1 621 + G-T MSDl 1.3 Al507 NBDl AF506 NBDl s5491 NBDl S549R NED1 A559T NED1 N1303K NBDP G551 D NBDl G551S NBDl G1244E NBDP S1255P NBDP G1349D NBDP RI 17H MSDI R334W MSDl R347P MSDl 0.5 67.2 Rare 0.3 Rare 1.a 2.4 Rare Rare Rare Rare 0.6 0.4 0.5 PI PI PI PI PI PI PI PI PS PI PI PI PS PS PS NED, nucleotide-binding domain; MSD, membrane-spanning domain; PI, pancreatic insufficiency; PS, pancreatic sufficiency. Login to comment
56 Some nucleotide-binding domain mutants (such as G551D) have very little function, in some (such as S1255P) ATP is less potent at stimulating activity, and the absolute activity of others (such as G551S, G1244E, and G1349D) is reduced (Anderson and Welsh, 1992; Drumm et al., 1991). Login to comment

[hide] The spectrum of cystic fibrosis mutations. Trends Genet. 1992 Nov;8(11):392-8. Tsui LC
The spectrum of cystic fibrosis mutations.
Trends Genet. 1992 Nov;8(11):392-8., [PMID:1279852]

Abstract [show]
Although the major mutation causing cystic fibrosis accounts for almost 70% of mutant chromosomes screened, almost 300 sequence alterations have been identified in the gene during the past two and a half years. At least 230 of these mutations are probably associated with disease. This rapid accumulation of data is in part due to the highly coordinated effort by members of the Cystic Fibrosis Genetic Analysis Consortium. The information is not only essential to genetic diagnosis, but also will aid in understanding the structure and function of the protein, and possibly in correlating genotype with phenotype.

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99 Most notable are the mutations identified at the glycine residues in the Walker motifs (regions highly conserved among ATP-binding proteins), namely, G458V and G551D in NBF1 (Refs 16, 10), and G1244E and G1349D at the corresponding residues in NBF2 (Refs 17, 18), suggesting that ATP binding is essential for CFTR function. Login to comment
123 8 NO. 11 m []~EVIEWS G551D R553Q G551S I L558S aI~7 S5491 I I 1&559T A455F E5040 I&F508 V520F SS49NII IIR560T PS74H I G458V G480C $492F /" • ss,9 II III* oa. / III / NBF1 ~t ~t NBF2 I I I I I III I I I 11234V G1244E IS1255P D1270N II I Q1291H N1303K G1349D S1251N W1282R] F1286S N1303H Q1283M, FIG[] Cystic fibrosis (missense) mutations located within the two presumptive ATP-binding domains (NBF1 and NBF2) of CFTR. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Br Med Bull. 1992 Oct;48(4):754-65. Higgins CF
Cystic fibrosis transmembrane conductance regulator (CFTR).
Br Med Bull. 1992 Oct;48(4):754-65., [PMID:1281034]

Abstract [show]
Since the identification of the CF gene, less than 3 years ago, progress in analysing the function of its product, the cystic fibrosis transmembrane conductance regulator (CFTR), has been remarkable. It is now clear that CFTR functions as a small conductance chloride channel in epithelial membranes. However, many other questions remain unanswered. How does a defect in this channel result in the various pathologies associated with cystic fibrosis? Does CFTR have additional functions? How do CF mutations alter the function of the protein? Tools are now available to address these and other questions. Many features of CFTR activity suggest that pharmacological interventions may be possible. Nevertheless, an enhanced understanding of CFTR function is still essential before this basic research will provide direct benefit to CF sufferers.

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82 First, a mutation which has been associated with the disease (G1349D), when introduced into CFTR does not appear to affect its ability to generate a chloride channel or alter the characteristics of that channel;3 this mutation might cause CF by altering another function of CFTR. Login to comment
100 This view is supported by the finding that one CF mutation (G1349D) does not appear to alter channel function,3 although it must be emphasised that other explanations for this can be advanced. Login to comment

[hide] Effects of cystic fibrosis and congenital bilatera... Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4. Mickle JE, Milewski MI, Macek M Jr, Cutting GR
Effects of cystic fibrosis and congenital bilateral absence of the vas deferens-associated mutations on cystic fibrosis transmembrane conductance regulator-mediated regulation of separate channels.
Am J Hum Genet. 2000 May;66(5):1485-95. Epub 2000 Apr 4., [PMID:10762539]

Abstract [show]
The protein defective in cystic fibrosis (CF), the CF transmembrane-conductance regulator (CFTR), functions as an epithelial chloride channel and as a regulator of separate ion channels. Although the consequences that disease-causing mutations have on the chloride-channel function have been studied extensively, little is known about the effects that mutations have on the regulatory function. To address this issue, we transiently expressed CFTR-bearing mutations associated with CF or its milder phenotype, congenital bilateral absence of the vas deferens, and determined whether mutant CFTR could regulate outwardly rectifying chloride channels (ORCCs). CFTR bearing a CF-associated mutation in the first nucleotide-binding domain (NBD1), DeltaF508, functioned as a chloride channel but did not regulate ORCCs. However, CFTR bearing disease-associated mutations in other domains retained both functions, regardless of the associated phenotype. Thus, a relationship between loss of CFTR regulatory function and disease severity is evident for NBD1, a region of CFTR that appears important for regulation of separate channels.

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50 Expression Analysis The mutations DF508, R1070W, D1270N, and G1349D were created in the vector pBQ4.7 containing CFTR cDNA (pBQ4.7 is a gift from J. Rommens and L. Login to comment
51 C. Tsui), by single-stranded mutagenesis (Youssoufian et al. 1995), and then were shuttled into pRSV-CFTR, a Rous sarcoma virus (RSV)-driven expression plasmid, by use of Kpn2I and HpaI (for DF508) or NcoI and SalI (for R1070W, D1270N, and G1349D) restriction sites common to both plasmids (Fulmer et al. 1995). Login to comment
75 Also selected for study were two mutations in NBF2-D1270N and G1349D, which are associated with different pulmonary phenotypes (table 1). Login to comment
77 For comparison purposes, we selected the CF-associated mutation G1349D, since it occurs in the same functional domain as does D1270N. Login to comment
97 OF CASES PHENOTYPE Lung Status Pancreatic Status Sweat Cl2 Fertility Normala Abnormal Not Reported Sufficient Insufficient Not Reported Reported (Mean 5 SEM [mmol/literb ]) Not Reported Subfertilec Not Reported R1070W (7)d 5 0 2 5 0 2e 6 ( ) 50.2 5 13.4 1 6 1 CBAVD R1070P (2)f 0 1 1 0 1 1 1 (Positive) 1 0 2 CF R1070Q (14)g 0 7 7 0 7 7 7 (Positive) 7 2 12 CF D1270N (9)h 4 0 5 4 0 5 3 ( ) 77.5 5 16.7 6 3 6 CBAVD G1349D (3)i 0 0 3 0 0 3 ) 3 1 2 CF a No history of chronic lung disease. Login to comment
134 The initial mutation report (Beaudet et al. 1991) indentified G1349D on two CF chromosomes, and, on the basis of these cases, G1349D has been described as a CF-associated mutation (Gregory et al. 1991; Anderson and Welsh 1992), with specific reference to PI (Welsh and Smith 1993). Login to comment
173 C, CBAVD(D1270N) and CF(G1349D) mutations in NBF2 had I-V profiles comparable to those of wild-type CFTR. Login to comment
180 Thus, the combination of I-V relationship and response to inhibitors allowed dissection of whole-cell Cl-currents into two components: outwardly rectified and DIDS sensitive, carried by separate channels such as ORCCs; and linear, DIDS insensitive, and gli- Table 2 Summary of Processing and Whole-Cell Function of CFTR Mutants DOMAIN AND MUTATION PHENOTYPE CFTR STATUS a Processingb Function Band A Band B Band C Cl2 Channel Regulatoryc Not applicable: Wild type Normal 2 2 111 111 1 NBF1:d A455Ee CFe 1 11 2 111 1 DF508 CF 1 11 2 1 2 G551D CF 2 2 111 1 2 TMD2: R1070W CBAVD 2 1 11 111 1 R1070P CF 1 11 2 111 1 R1070Q CF 2 1 11 111 1 NBF2: D1270N CBAVD 2 2 111 111 1 G1349D CF 2 2 111 111 1 a A minus sign (2) denotes absence; a single plus sign (1) denotes "low"; a double plus sign (11) denotes "intermediate"; and a triple plus sign denotes "high." Login to comment
190 Likewise, cells expressing either of the NBF2 mutants- D1270N (CBAVD) and G1349D (CF)-had both components of whole-cell Cl2 currents (fig. 3C). Login to comment
214 Of particular note are the results obtained with the NBF2 mutation G1349D. Login to comment
215 The latter mutation is comparable, in terms of nature (glycine to aspartic acid) and location (Walker C motif), to the NBF1 mutation G551D; yet, CFTR bearing G1349D-generated DIDS-sensitive currents that were attributed to the regulation of ORCCs. Login to comment

[hide] Cystic fibrosis: the 'bicarbonate before chloride'... Curr Biol. 2001 Jun 26;11(12):R463-6. Wine JJ
Cystic fibrosis: the 'bicarbonate before chloride' hypothesis.
Curr Biol. 2001 Jun 26;11(12):R463-6., [PMID:11448786]

Abstract [show]
The specific effects of some mutations that cause cystic fibrosis suggest that reduced HCO(3)(-) transport is the key to understanding cystic fibrosis pathology. But there is a puzzling discrepancy between measures of CFTR-mediated chloride conductance in expression systems and the sweat chloride values of patients.

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52 Ion transport (% WT) 42 41 69 75 >100 >100 98 + 103 100 + + 120 Pancreatic sufficient Pancreatic insufficient Bicarbonate Chloride - intermediate Chloride - high Unknown WT D648V R117H R1070Q H949Y G551S H620Q I148T A1067T G178R G970R S1255P G1244E G551D G1349D 0 0.5 1 1.5 2 2.5 Current Biology ࢞F508 Dispatch R absence of the vas deferens [16]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Neuron. 1992 May;8(5):821-9. Welsh MJ, Anderson MP, Rich DP, Berger HA, Denning GM, Ostedgaard LS, Sheppard DN, Cheng SH, Gregory RJ, Smith AE
Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation.
Neuron. 1992 May;8(5):821-9., [PMID:1375035]

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225 For example, expression of CFTR-G1349D, containing a mutation in NBD2, generates a completely glycosylated protein and CAMP-regulated Cl-channels (Gregory et al., 1991). Login to comment

[hide] Altered channel gating mechanism for CFTR inhibiti... FEBS Lett. 2004 Jan 30;558(1-3):52-6. Taddei A, Folli C, Zegarra-Moran O, Fanen P, Verkman AS, Galietta LJ
Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker.
FEBS Lett. 2004 Jan 30;558(1-3):52-6., [PMID:14759515]

Abstract [show]
The thiazolidinone CFTR(inh)-172 was identified recently as a potent and selective blocker of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. Here, we characterized the CFTR(inh)-172 inhibition mechanism by patch-clamp and short-circuit analysis using cells stably expressing wild-type and mutant CFTRs. CFTR(inh)-172 did not alter CFTR unitary conductance (8 pS), but reduced open probability by >90% with K(i) approximately 0.6 microM. This effect was due to increased mean channel closed time without changing mean channel open time. Short-circuit current experiments indicated similar CFTR(inh)-172 inhibitory potency (K(i) approximately 0.5 microM) for inhibition of Cl(-) current in wild-type, G551D, and G1349D CFTR; however, K(i) was significantly reduced to 0.2 microM for DeltaF508 CFTR. Our studies provide evidence for CFTR inhibition by CFTR(inh)-172 by a mechanism involving altered CFTR gating.

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4 Short-circuit current experiments indicated similar CFTRinh-172 inhibitory potency (KiW W0.5 W WM) for inhibition of Cl3 current in wild-type, G551D, and G1349D CFTR; however, Ki was signi'cantly reduced to 0.2 W WM for v vF508 CFTR. Login to comment
93 To test whether CFTRinh-172 interacts with one of the NBDs, we compared the inhibitory potency of CFTRinh-172 on wild-type CFTR and the mutants G551D and G1349D that produce defective CFTR gating NBD-1 and NBD-2, respectively. Login to comment
96 The &#a3;avone genistein (at 200 WM) together with cpt-cAMP was used to maximally activate the CFTR mutants G551D and G1349D. Login to comment
97 CFTRinh-172 blocked G551D and G1349D Cl3 currents with potency not signi'cantly di&#a1;erent from that for inhibition of wild-type CFTR, with Ki of 0.53 WM (nH = 0.90) and 0.51 (nH = 1.17), respectively, for G551D and G1349D (Fig. 4B,C). Login to comment
114 A: Representative short-circuit current experiments on permeabilized FRT cells expressing wild-type CFTR, or G551D, G1349D, and vF508 mutants. Login to comment
115 cpt-cAMP concentration was 100 WM and genistein concentrations were 50 WM (wild-type and vF508) or 200 WM (G551D and G1349D). Login to comment
121 After genistein activation, the CFTRinh-172 inhibitory potency did not di&#a1;er signi'cantly (Ki = 0.48; nH = 0.96) from that measured for G551D and G1349D. Login to comment
124 Interestingly, vF508-CFTR Cl3 currents were inhibited by CFTRinh-172 with a signi'cant about twofold greater e/cacy than found for wild-type CFTR and the G551D and G1349D mutants (Fig. 4B,C). Login to comment
143 We also tested the e&#a1;ect of CFTR mutations that impair NBD function and thus reduce CFTR opening, including G551D and G1349D. Login to comment
145 CFTRinh-172 had comparable inhibitory potency for reducing Cl3 currents for wild-type CFTR, G551D, and G1349D. Login to comment

[hide] Molecular targeting of CFTR as a therapeutic appro... Trends Pharmacol Sci. 2007 Jul;28(7):334-41. Epub 2007 Jun 18. Amaral MD, Kunzelmann K
Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis.
Trends Pharmacol Sci. 2007 Jul;28(7):334-41. Epub 2007 Jun 18., [PMID:17573123]

Abstract [show]
One of the major challenges facing the pharmaceutical field is the identification of novel, 'druggable' targets common to distinct diseases that, despite their clinical diversity, share the same basic molecular defect(s) - thus, being termed 'horizontal diseases'. Membrane proteins constitute one of the largest families in the human genome and, given their major roles in cells and organisms, they are relevant to common human disorders such as cardiovascular disease and cancer, but also to rare genetic conditions such as cystic fibrosis (CF). Here, we review therapeutic approaches to correcting the basic defect in CF, which is caused mainly by the intracellular retention of a misfolded protein, and focus on various recent drug-discovery strategies for this important and paradigmatic disease. These strategies have possible applications in many membrane protein disorders, including other channelopathies. The mechanisms of action of potent and specific compounds, representing promising drug leads for CF pharmacotherapy, are explained and discussed.

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135 Phenylglycines and sulfonamides (potencies >100 nM) improve F508del-CFTR, G551D-CFTR and G1349D-CFTR gating, but only after cAMP activation [48,49]. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Drugs. 2010 Feb 12;70(3):241-59. doi: 10.2165/11316160-000000000-00000. Becq F
Cystic fibrosis transmembrane conductance regulator modulators for personalized drug treatment of cystic fibrosis: progress to date.
Drugs. 2010 Feb 12;70(3):241-59. doi: 10.2165/11316160-000000000-00000., [PMID:20166764]

Abstract [show]
This article considers the issue of personalized drug discovery for the orphan disease cystic fibrosis (CF) to deliver a candidate for therapeutic development. CF is a very complicated disease due to numerous anomalies of the gene leading to progressive severity and morbidity. Despite extensive research efforts, 20 years after the cloning of the CF gene, CF patients are still waiting for a curative treatment as prescribed medications still target the secondary manifestations of the disease rather than the gene or the CF transmembrane conductance regulator (CFTR) protein. New therapeutics aimed at improving mutant CFTR functions, also known as 'protein repair therapy' are nevertheless hoped and predicted to replace some of the currently used therapy, while improving the quality of life as well as life expectancy of CF patients. Although there is substantial variability in the cost of treating CF between countries, a protein repair therapy should also alleviate the financial burden of medical costs for CF patients and their families. Finding new drugs or rediscovering old ones for CF is critically dependent on the delivery of molecular and structural information on the CFTR protein, on its mutated version and on the network of CFTR-interacting proteins. The expertise needed to turn compounds into marketable drugs for CF will depend on our ability to provide biological information obtained from pertinent models of the disease and on our success in transferring safe molecules to clinical trials. Predicting a drug-induced response is also an attractive challenge that could be rapidly applied to patients.

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102 [28,35] For class III mutants (e.g. G551D, G1349D), no or reduced activation of the CFTR chloride function at the plasma membrane also leads to epithelial Cl- impermeability and to a severe disease as for the first two categories. Login to comment
209 [76] Similar effects were observed for G551D, G1349D and D1152H mutants. Login to comment

[hide] Emergent properties of proteostasis in managing cy... Cold Spring Harb Perspect Biol. 2011 Feb 1;3(2). pii: a004499. doi: 10.1101/cshperspect.a004499. Balch WE, Roth DM, Hutt DM
Emergent properties of proteostasis in managing cystic fibrosis.
Cold Spring Harb Perspect Biol. 2011 Feb 1;3(2). pii: a004499. doi: 10.1101/cshperspect.a004499., [PMID:21421917]

Abstract [show]
Cystic fibrosis (CF) is a consequence of defective recognition of the multimembrane spanning protein cystic fibrosis conductance transmembrane regulator (CFTR) by the protein homeostasis or proteostasis network (PN) (Hutt and Balch (2010). Like many variant proteins triggering misfolding diseases, mutant CFTR has a complex folding and membrane trafficking itinerary that is managed by the PN to maintain proteome balance and this balance is disrupted in human disease. The biological pathways dictating the folding and function of CFTR in health and disease are being studied by numerous investigators, providing a unique opportunity to begin to understand and therapeutically address the role of the PN in disease onset, and its progression during aging. We discuss the general concept that therapeutic management of the emergent properties of the PN to control the energetics of CFTR folding biology may provide significant clinical benefit.

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38 Proteostasis in Managing Cystic Fibrosis Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004499 3 Cold Spring Harbor Laboratory Press at SEMMELWEIS UNIV OF MEDICINE on December 5, corrector activity PC/PR potentiator activity PC/PR ER Golgi Lysosome Endosomes Native structure Native structure Apical surface C B SDS-PAGE ƊF508 ƊF508 Ɗ F 5 0 8 w t ERAD Wild-type WT A G551D (NBD1)/G1349D (NBD2) traffic to cell surface but lack channel activity RCN P23 HSTF1 HSP47 HSPA1L CYPB PPIA COC37 HOP Hsp40 HSP70 CFTR HSP90 Core B CANX CHIP CCT3 CCT4 USP49 GRP75 CCT5 CCT1 DNAJA2 BAG1 HSP60 HSP105 HSP21 HSP22 BAG2 RCN2 BAG2 HSC70 GRP78 UBC UBB Aha1 Regulatory Figure 3. Login to comment
40 (A) Illustrated is the trafficking itinerary of WTand three vCFTR (DF508, G551E, G1349D) through the exocytic pathway. Login to comment
47 The G551E and G1349D mutants (purple) are folded and traffic normally to cell surface. Login to comment
49 The G551E and G1349D vCFTR only require a potentiator to open the channel and restore function. Login to comment
145 Moreover, a recent comparison of 13 potentiators showed that three different mutants, including DF508 (NBD1, ER degraded, Fig. 3A), G551D (NBD1, cell surface localized, Fig. 3A), and G1349D (NBD2, cell surface localized) (Pedemonte et al. 2010), expressed in FRT cells or the human alveolar epithelial cells A549 responded to 10 of the 13 compounds tested, supporting the conclusion that these potentiators may be binding to vCFTR directly and their binding is not influenced by cellular environment (Pedemonte et al. 2010). Login to comment

[hide] Stimulation of Wild-Type, F508del- and G551D-CFTR ... Front Pharmacol. 2011 Aug 23;2:48. doi: 10.3389/fphar.2011.00048. eCollection 2011. Dannhoffer L, Billet A, Jollivet M, Melin-Heschel P, Faveau C, Becq F
Stimulation of Wild-Type, F508del- and G551D-CFTR Chloride Channels by Non-Toxic Modified pyrrolo[2,3-b]pyrazine Derivatives.
Front Pharmacol. 2011 Aug 23;2:48. doi: 10.3389/fphar.2011.00048. eCollection 2011., [PMID:21897819]

Abstract [show]
Cystic fibrosis (CF) is a major inherited disorder involving abnormalities of fluid and electrolyte transport in a number of different organs due to abnormal function of cystic fibrosis transmembrane conductance regulator (CFTR) protein. We recently identified a family of CFTR activators, which contains the hit: RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine]. Here, we further evaluated the effect of the chemical modifications of the RP107-OH radical on CFTR activation. The replacement of the OH radical by a fluorine atom at position 2 (RP193) or 4 (RP185) significantly decreased the toxicity of the compounds without altering the ability to activate CFTR, especially for RP193. The non-toxic compound RP193 has no effect on cAMP production but stimulates the channel activity of wild-type CFTR in stably transfected CHO cells, in human bronchial epithelial NuLi-1 cells, and in primary culture of human bronchial epithelial cells (HBEC). Whole-cell and single patch-clamp recordings showed that RP193 induced a linear, time- and voltage-independent current, which was fully inhibited by two different and selective CFTR inhibitors (CFTRinh-172 and GP(inh)5a). Moreover, RP193 stimulates CFTR in temperature-rescued CuFi-1 (F508del/F508del) HBEC and in CHO cells stably expressing G551D-CFTR. This study shows that it is feasible to reduce cytotoxicity of chemical compounds without affecting their potency to activate CFTR and to rescue the class 2 F508del-CFTR and class 3 G551D-CFTR CF mutant activities.

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20 Patients with a class 3 mutation of CFTR (e.g.,G551D,G1349D) have also a severe form of CF, because they do not have or reduced activation of the CFTR chloride function at the plasma membrane. Login to comment

[hide] [Mucoviscidosis: CFTR mutation-specific therapy: a... Arch Pediatr. 2013 Jan;20(1):63-73. doi: 10.1016/j.arcped.2012.10.018. Epub 2012 Nov 27. Leonard A, Leal T, Lebecque P
[Mucoviscidosis: CFTR mutation-specific therapy: a ray of sunshine in a cloudy sky].
Arch Pediatr. 2013 Jan;20(1):63-73. doi: 10.1016/j.arcped.2012.10.018. Epub 2012 Nov 27., [PMID:23199563]

Abstract [show]
There is a need to find a cure for pulmonary disease in cystic fibrosis (CF), though full benefit of this approach will be restricted to those patients with well-preserved lungs. The most promising route is currently that of a pharmacological mutation-specific approach aiming at correcting the mechanism by which mutations lead to impairment of chloride conductance across respiratory epithelial cells. In the past 14years, 7 candidate drugs (CPX, 4PBA, gentamicin, PTC124, VX-770 or Ivacaftor, VX-809 or Lumacaftor, and Miglustat) have been investigated in CF patients. A postulate of 14 out of the 15 published studies has been that an effective agent had to improve total chloride secretion as assessed in vivo by nasal potential difference measurements. The present review casts a critical look at these studies. Apparent inconsistencies are discussed as well as possible limitations of nasal potential difference measurements as outcome parameters in these trials. Primarily targeting a mutation carried by less than 2% of French CF patients, the 2 Ivacaftor studies could well be a milestone on the long road toward a cure for CF. However, further data on safety and long-term efficacy are obviously needed and the current price of this medication in the US would make it unaffordable for European patients.

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178 De re &#b4;centes donne &#b4;es in vitro sugge `rent que cette me &#b4;dication soit e &#b4;galement efficace sur 9 autres mutations de la me c6;me classe (G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, G1349D) [50,51]. Login to comment

[hide] Vx-770 potentiates CFTR function by promoting deco... Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25. Jih KY, Hwang TC
Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle.
Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):4404-9. doi: 10.1073/pnas.1215982110. Epub 2013 Feb 25., [PMID:23440202]

Abstract [show]
Vx-770 (Ivacaftor), a Food and Drug Administration (FDA)-approved drug for clinical application to patients with cystic fibrosis (CF), shifts the paradigm from conventional symptomatic treatments to therapeutics directly tackling the root of the disease: functional defects of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel caused by pathogenic mutations. The underlying mechanism for the action of Vx-770 remains elusive partly because this compound not only increases the activity of wild-type (WT) channels whose gating is primarily controlled by ATP binding/hydrolysis, but also improves the function of G551D-CFTR, a disease-associated mutation that abolishes CFTR's responsiveness to ATP. Here we provide a unified theory to account for this dual effect of Vx-770. We found that Vx-770 enhances spontaneous, ATP-independent activity of WT-CFTR to a similar magnitude as its effects on G551D channels, a result essentially explaining Vx-770's effect on G551D-CFTR. Furthermore, Vx-770 increases the open time of WT-CFTR in an [ATP]-dependent manner. This distinct kinetic effect is accountable with a newly proposed CFTR gating model depicting an [ATP]-dependent "reentry" mechanism that allows CFTR shuffling among different open states by undergoing multiple rounds of ATP hydrolysis. We further examined the effect of Vx-770 on R352C-CFTR, a unique mutant that allows direct observation of hydrolysis-triggered gating events. Our data corroborate that Vx-770 increases the open time of WT-CFTR by stabilizing a posthydrolytic open state and thereby fosters decoupling between the gating cycle and ATP hydrolysis cycle. The current study also suggests that this unique mechanism of drug action can be further exploited to develop strategies that enhance the function of CFTR.

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271 14. Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment
334 Cai Z, Taddei A, Sheppard DN (2006) Differential sensitivity of the cystic fibrosis (CF)- associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment

[hide] Converting nonhydrolyzable nucleotides to strong c... J Biol Chem. 2013 Jun 14;288(24):17122-33. doi: 10.1074/jbc.M112.442582. Epub 2013 Apr 25. Okeyo G, Wang W, Wei S, Kirk KL
Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations.
J Biol Chem. 2013 Jun 14;288(24):17122-33. doi: 10.1074/jbc.M112.442582. Epub 2013 Apr 25., [PMID:23620589]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the only ligand-gated ion channel that hydrolyzes its agonist, ATP. CFTR gating has been argued to be tightly coupled to its enzymatic activity, but channels do open occasionally in the absence of ATP and are reversibly activated (albeit weakly) by nonhydrolyzable nucleotides. Why the latter only weakly activates CFTR is not understood. Here we show that CFTR activation by adenosine 5'-O-(thiotriphosphate) (ATPgammaS), adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) is enhanced substantially by gain of function (GOF) mutations in the cytosolic loops that increase unliganded activity. This enhancement correlated with the base-line nucleotide-independent activity for several GOF mutations. AMP-PNP or ATPgammaS activation required both nucleotide binding domains (NBDs) and was disrupted by a cystic fibrosis mutation in NBD1 (G551D). GOF mutant channels deactivated very slowly upon AMP-PNP or ATPgammaS removal (taudeac approximately 100 s) implying tight binding between the two NBDs. Despite this apparently tight binding, neither AMP-PNP nor ATPgammaS activated even the strongest GOF mutant as strongly as ATP. ATPgammaS-activated wild type channels deactivated more rapidly, indicating that GOF mutations in the cytosolic loops reciprocally/allosterically affect nucleotide occupancy of the NBDs. A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPgammaS, indicating that these analogs interact differently with the NBDs. We conclude that poorly hydrolyzable nucleotides are less effective than ATP at opening CFTR channels even when they bind tightly to the NBDs but are converted to stronger agonists by GOF mutations.

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13 A GOF mutation substantially rescued defective ATP-dependent gating of G1349D-CFTR, a cystic fibrosis NBD2 signature sequence mutant. Login to comment
14 Interestingly, the G1349D mutation strongly disrupted activation by AMP-PNP but not by ATPॹS, indicating that these analogs interact differently with the NBDs. Login to comment
166 The analogous mutation in the NBD2 signature sequence (G1349D) causes milder CF disease apparently because it incompletely disrupts channel gating. Login to comment
167 G1349D-CFTR channels retain ATP-dependent activity but with a maximal single chan- FIGURE 3. Login to comment
180 As reported by others (12), we detected relatively small ATP-dependent control currents for G1349D-CFTR channels in excised macropatches. Login to comment
182 Introduction of one of the GOF mutations into this CF mutant (K978C/G1349D-CFTR) substantially rescued its activity as evidenced by much higher ATP-dependent control currents and much lower relative activation by the potentiators (Fig. 6, B and C). Login to comment
183 K978C/G1349D-CFTR channels also exhibited detectable currents in the absence of any bath nucleotide as expected (Fig. 6D). Login to comment
184 Unlike K978C/G551D-CFTR channels, K978C/G1349D-CFTR channels remained sensitive to ATPॹS as did the G1349D mutant without the GOF substitution (Fig. 6, D-F). Login to comment
186 The G1349D results support two conFIGURE 4. Login to comment
203 First, the defective ATP-dependent gating of G1349D-CFTR channels can be rescued substantially by a GOF mutation located in the cytosolic loops well away from the NBDs. Login to comment
205 The P-N-P linkage in the latter may reduce its ability to accommodate to distortions in the nucleotide binding pockets and dimer interface that are induced by the G1349D mutation. Login to comment
236 Rescue of G1349D-CFTR gating by K978C and differential activation of this NBD2 mutant by ATPॹS and AMP-PNP. Login to comment
237 A, a representative macropatch record shows low control current for G1349D-CFTR (1.5 mM ATP) and large activation by serial addition of 10 òe;M 5-nitro-2-(3-phenylpropylamino) benzamide (NPPB-AM) and 30 òe;M curcumin. Login to comment
238 B, a corresponding macropatch record for K978C/G1349D-CFTR shows large control current and relatively small activation by NPPB-AM and curcumin. Login to comment
239 C, shown are mean control currents (1.5 mM ATP) and mean -fold activation by the combination of curcumin and NPPB-AM for G1349D-CFTR and K978C/G1349D-CFTR (n afd; 6-17; **, p b0d; 0.01 compared with G1349D). Login to comment
240 D and E, representative macropatch records show much smaller activation of K978C/G1349D-CFTR or G1349D-CFTR current by 2 mM AMP-PNP versus 1.5 mM ATPॹS. Hex, hexokinase. Login to comment
241 F, mean relative activation of G1349D-CFTRandK978C/G1349D-CFTRbyAMP-PNPandATPॹS.Percentstimulationwascalculatedbynormalizingtheincreaseincurrentabovebaseline(i.e. current in the absence of nucleotide) to the control current before ATP removal by hexokinase/glucose. Login to comment
285 GOF Mutations Rescue G551D and G1349D CF Channels but in Different Ways-The ABC signature sequences line the two ATP binding pockets and play important roles in ABC transporter function and CFTR channel gating. Login to comment
288 The analogous mutation in NBD2 (G1349D) causes less severe disease because it incompletely inhibits ATP-dependent gating (12). Login to comment
291 In contrast, we observed a more authentic rescue for the G1349D- K978C construct in the form of much greater ATP-dependent currents and lower relative activation by potentiators. Login to comment
293 This more authentic rescue of the G1349D channel presumably relates to the incomplete loss of ATP-dependent gating caused by this signature sequence mutation versus the complete abolition of ATP dependence by the corresponding G551D mutation (12). Login to comment
294 G1349D-CFTR rescue by the K978C GOF mutation is analogous to its enhancement of CFTR activation by ATPॹS or AMP-PNP. Login to comment
297 In the case of G1349D-CFTR, the signature sequence mutation in NBD2 may compromise the efficacy of ATP to open the channel. Login to comment

[hide] Managing the underlying cause of cystic fibrosis: ... Paediatr Drugs. 2013 Oct;15(5):393-402. doi: 10.1007/s40272-013-0035-3. Galietta LJ
Managing the underlying cause of cystic fibrosis: a future role for potentiators and correctors.
Paediatr Drugs. 2013 Oct;15(5):393-402. doi: 10.1007/s40272-013-0035-3., [PMID:23757197]

Abstract [show]
Cystic fibrosis (CF), a severe genetic disease, is caused by mutations that alter the structure and function of CFTR, a plasma membrane channel permeable to chloride and bicarbonate. Defective anion transport in CF irreversibly damages the lungs, pancreas, liver, and other organs. CF mutations cause loss of CFTR function in multiple ways. In particular, class 3 mutations such as p.Gly551Asp strongly decrease the time spent by CFTR in the open state (gating defect). Instead, class 2 mutations impair the maturation of CFTR protein and its transport from the endoplasmic reticulum to the plasma membrane (trafficking defect). The deletion of phenylalanine 508 (p.Phe508del), the most frequent mutation among CF patients (70-90 %), destabilizes the CFTR protein, thus causing both a trafficking and a gating defect. These two defects can be overcome with drug-like molecules generically called correctors and potentiators, respectively. The potentiator Kalydeco (also known as Ivacaftor or VX-770), developed by Vertex Pharmaceuticals, has been recently approved by the US FDA and the European Medicines Agency (EMA) for the treatment of CF patients carrying at least one CFTR allele with the p.Gly551Asp mutation (2-5 % of all patients). In contrast, the corrector VX-809, which significantly improves p.Phe508del-CFTR trafficking in vitro, is still under study in clinical trials. Because of multiple defects caused by the p.Phe508del mutation, it is probable that rescue of the mutant protein will require combined treatment with correctors having different mechanisms of action. This review evaluates the status of experimental and clinical research in pharmacotherapy for the CF basic defect.

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103 In particular, class 3 mutations such as p.Gly551Asp and p.Gly1349Asp, which cause a gating defect even more severe than that of p.Phe508del but no trafficking problems, are highly responsive to genistein and other potentiators [19, 39, 40]. Login to comment
108 There are several studies describing the discovery of very effective potentiators, in many cases with nanomolar potency, that can rescue channel activity not only of p.Phe508del, but also of p.Gly551Asp, p.Gly1349Asp, p.Glu193Lys, p.Gly970Arg, p.Asp1152His, and other mutations [51, 52]. Login to comment

[hide] A comprehensive assay for CFTR mutational analysis... Clin Chem. 2013 Oct;59(10):1481-8. doi: 10.1373/clinchem.2013.206466. Epub 2013 Jun 17. Abou Tayoun AN, Tunkey CD, Pugh TJ, Ross T, Shah M, Lee CC, Harkins TT, Wells WA, Tafe LJ, Amos CI, Tsongalis GJ
A comprehensive assay for CFTR mutational analysis using next-generation sequencing.
Clin Chem. 2013 Oct;59(10):1481-8. doi: 10.1373/clinchem.2013.206466. Epub 2013 Jun 17., [PMID:23775370]

Abstract [show]
BACKGROUND: Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS: We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine (PGM) platform. RESULTS: The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500x to 3500x, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS: With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.

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53 of cases af9;/af9; c.1521_1523delCTT; c.1521_1523delCTT èc;F508; èc;F508 CF Yes 97 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.350Gb0e;A èc;F508; R117H CF Yes 53; 50 Dartmouth 1 af9;/af9; c.350Gb0e;A; c.1477Cb0e;T R117H; Q493*b CF Yes 52; 49 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.1000Cb0e;T èc;F508; R334W CF Yes 49; 54 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.489af9;1Gb0e;T èc;F508; 621af9;1Gb0e;T CF Yes 48; 47 Dartmouth 1 af9;/af9; c.1521_1523delCTT; c.1364Cb0e;A èc;F508; A455E CF Yes 51; 46 Dartmouth 1 af9;/af9; c.489af9;1Gb0e;T; c.2988af9;1Gb0e;A 621af9;1Gb0e;T; 3120af9;1Gb0e;A CF Yes 48; 49 Coriell 1 af9;/af9; c.1521_1523delCTT; c.1657Cb0e;T èc;F508; R553* CF Yes 44; 49c Coriell 2 af9;/af9; c.1521_1523delCTT; c.3528delC èc;F508; 3659delC CF Yes 46; 46 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.579af9;1Gb0e;T 621af9;1Gb0e;T; 711af9;1Gb0e;T CF Yes 50; 51 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.254Gb0e;A 621af9;1Gb0e;T; G85E CF Yes 50; 45 Coriell 1 af9;/af9; c.1521_1523delCTT; c.1679Gb0e;C èc;F508; R560T CF Yes 44; 52 Coriell 1 af9;/af9; c.489af9;1Gb0e;T; c.1364Cb0e;A 621af9;1Gb0e;T; A455E CF Yes 50; 49 Coriell 1 af9;/af9; c.3909Cb0e;G; c.4046Gb0e;A N1303K; G1349D CF Yes 47; 52 Coriell 1 af9;/af9; c.2657af9;5Gb0e;A; c.2657af9;5Gb0e;A 2789af9;5Gb0e;A; 2789af9;5Gb0e;A CF Yes 100 Coriell 1 af9;/af9; c.1040Gb0e;C; c.1652Gb0e;A R347P; G551D CF Yes 51; 49 Coriell 1 af9;/af9; c.1000Cb0e;T; c.3368-2Ab0e;T R334W; 3500-2Ab0e;G CF Yes 53; 45 Coriell 1 af9;/af9; c.254Gb0e;A; c.3454Gb0e;C G85E; D1152H CF Yes 44; 47 Coriell 1 af9;/af9; c.1521_1523delCTT; c.350Gb0e;A èc;F508; R117H CF Yes 49; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.54-5940_273af9;10250del21kb èc;F508; CFTRdel2,3 CF Yes 47; N/Ad Coriell 1 af9;/af9; c.1521_1523delCTT; c.1766af9;1Gb0e;A èc;F508; 1898af9;1Gb0e;A CF Yes 47; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.2051_2052delAAinsG èc;F508; K684Sfs CF Yes 47; 50 Coriell 1 af9;/af9; c.1521_1523delCTT; c.2052del èc;F508; K684Nfs*38 CF Yes 51; 55 Coriell 1 af9;/afa; c.1521_1523delCTT èc;F508 Carrier Yes 50c Dartmouth 16 af9;/afa; c.1652Gb0e;A G551D Carrier Yes 50c Dartmouth 5 af9;/afa; c.1519_1521delATC èc;I507 Carrier Yes 46 Dartmouth 1 af9;/afa; c.3454Gb0e;C D1152H Carrier Yes 50 Dartmouth 1 af9;/afa; c.1657Cb0e;T R553* Carrier Yes 51 Dartmouth 1 af9;/afa; c.178Gb0e;T E60* Carrier Yes 51 Dartmouth 1 af9;/afa; c.3846Gb0e;A W1282* Carrier Yes 45c Dartmouth 3 af9;/afa; c.1000Cb0e;T R334W Carrier Yes 51 Dartmouth 1 af9;/afa; c.1624Gb0e;T G542* Carrier Yes 47c Dartmouth 4 af9;/afa; c.3484Cb0e;T R1162* Carrier Yes 43 Dartmouth 1 af9;/afa; c.1766af9;1Gb0e;A 1898af9;1Gb0e;A Carrier Yes 57 Dartmouth 1 af9;/afa; c.3773_3774insT 3905insT (L1258Ffs*7) Carrier Yes 37 Dartmouth 1 af9;/afa; c.350Gb0e;A R117H Carrier Yes 50c Dartmouth 3 af9;/afa; c.1645Ab0e;C S549R Ab0e;C Carrier No N/A Dartmouth 1 af9;/afa; c.1040Gb0e;A R347H Carrier Yes 47 Dartmouth 1 af9;/afa; c.3909Cb0e;G N1303K Carrier Yes 46 Dartmouth 1 af9;/afa; c.3718-2477Cb0e;T 3849af9;10kbCb0e;T Carrier Yes 51 Coriell 1 af9;/afa; c.2988af9;1Gb0e;A 3120af9;1Gb0e;A Carrier Yes 49 Coriell 1 af9;/afa; c.489af9;1Gb0e;T 621af9;1Gb0e;T Carrier Yes 50 Coriell 1 af9;/afa; c.1585-1Gb0e;A 1717-1Gb0e;A Carrier Yes 51 Coriell 1 afa;/afa;e N/Af N/A Normal N/A N/A Dartmouth 9 a af9;/af9;, 2 pathogenic mutations; af9;/afa;, carrier of a single pathogenic mutation; afa;/afa;, absence of any pathogenic mutations. Login to comment

[hide] Effect of ivacaftor on CFTR forms with missense mu... J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23. Van Goor F, Yu H, Burton B, Hoffman BJ
Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
J Cyst Fibros. 2014 Jan;13(1):29-36. doi: 10.1016/j.jcf.2013.06.008. Epub 2013 Jul 23., [PMID:23891399]

Abstract [show]
BACKGROUND: Ivacaftor (KALYDECO, VX-770) is a CFTR potentiator that increased CFTR channel activity and improved lung function in patients age 6 years and older with CF who have the G551D-CFTR gating mutation. The aim of this in vitro study was to evaluate the effect of ivacaftor on mutant CFTR protein forms with defects in protein processing and/or channel function. METHODS: The effect of ivacaftor on CFTR function was tested in electrophysiological studies using a panel of Fischer rat thyroid (FRT) cells expressing 54 missense CFTR mutations that cause defects in the amount or function of CFTR at the cell surface. RESULTS: Ivacaftor potentiated multiple mutant CFTR protein forms that produce functional CFTR at the cell surface. These included mutant CFTR forms with mild defects in CFTR processing or mild defects in CFTR channel conductance. CONCLUSIONS: These in vitro data indicated that ivacaftor is a broad acting CFTR potentiator and could be used to help stratify patients with CF who have different CFTR genotypes for studies investigating the potential clinical benefit of ivacaftor.

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28 These include the most common CFTR gating mutation, G551D, as well as the G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D mutations [12]. Login to comment

[hide] Ivacaftor: a review of its use in patients with cy... Drugs. 2013 Sep;73(14):1595-604. doi: 10.1007/s40265-013-0115-2. Deeks ED
Ivacaftor: a review of its use in patients with cystic fibrosis.
Drugs. 2013 Sep;73(14):1595-604. doi: 10.1007/s40265-013-0115-2., [PMID:24030637]

Abstract [show]
Ivacaftor (Kalydeco) is a potentiator of the cystic fibrosis transmembrane conductance regulator (CFTR) and is the first drug that treats an underlying cause of cystic fibrosis to be licensed for use. Ivacaftor increases the open probability (i.e. gating) of CFTR channels with the G551D mutation, thus enhancing chloride transport, and is indicated in a number of countries for the treatment of cystic fibrosis in patients aged >/=6 years who carry this mutation. This review focuses on pharmacological, clinical efficacy and tolerability data relevant to the use of ivacaftor in this indication. In two 48-week, double-blind, phase III trials in patients aged >/=12 (STRIVE) or 6-11 (ENVISION) years with cystic fibrosis and the G551D mutation, oral ivacaftor 150 mg every 12 h significantly improved lung function relative to placebo, when used in combination with standard care. Significant improvements in pulmonary exacerbation risk (in STRIVE) as well as bodyweight and some aspects of health-related quality of life (both studies) were also seen with the drug versus placebo. Moreover, the beneficial effects of ivacaftor on parameters such as lung function and bodyweight were maintained over up to 96 weeks of treatment in an ongoing open-label extension of these studies. Ivacaftor was generally well tolerated, with headache, oropharyngeal pain, upper respiratory tract infection and nasal congestion being among the most common adverse events. Thus, ivacaftor expands the current treatment options for patients with cystic fibrosis who have the G551D mutation. Its potential for use in patients with other CFTR mutations is also of interest.

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35 For example, in rodent cells expressing G551D/S, G178R, S549N/R, G970R, G1244E, S1251N, S1255P or G1349D CFTR, ivacaftor increased channel open probability from B5 % of normal at baseline to 30-118 % of normal and increased chloride transport C16- fold (EC50 124-594 nmol/L). Login to comment

[hide] Catalyst-like modulation of transition states for ... J Gen Physiol. 2014 Feb;143(2):269-87. doi: 10.1085/jgp.201311089. Epub 2014 Jan 13. Csanady L, Torocsik B
Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating.
J Gen Physiol. 2014 Feb;143(2):269-87. doi: 10.1085/jgp.201311089. Epub 2014 Jan 13., [PMID:24420771]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is the chloride ion channel mutated in cystic fibrosis (CF) patients. It is an ATP-binding cassette protein, and its resulting cyclic nonequilibrium gating mechanism sets it apart from most other ion channels. The most common CF mutation (DeltaF508) impairs folding of CFTR but also channel gating, reducing open probability (Po). This gating defect must be addressed to effectively treat CF. Combining single-channel and macroscopic current measurements in inside-out patches, we show here that the two effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on CFTR, pore block and gating stimulation, are independent, suggesting action at distinct sites. Furthermore, detailed kinetic analysis revealed that NPPB potently increases Po, also of DeltaF508 CFTR, by affecting the stability of gating transition states. This finding is unexpected, because for most ion channels, which gate at equilibrium, altering transition-state stabilities has no effect on Po; rather, agonists usually stimulate by stabilizing open states. Our results highlight how for CFTR, because of its unique cyclic mechanism, gating transition states determine Po and offer strategic targets for potentiator compounds to achieve maximal efficacy.

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354 G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] The relative frequency of CFTR mutation classes in... J Cyst Fibros. 2014 Jul;13(4):403-9. doi: 10.1016/j.jcf.2013.12.003. Epub 2014 Jan 16. De Boeck K, Zolin A, Cuppens H, Olesen HV, Viviani L
The relative frequency of CFTR mutation classes in European patients with cystic fibrosis.
J Cyst Fibros. 2014 Jul;13(4):403-9. doi: 10.1016/j.jcf.2013.12.003. Epub 2014 Jan 16., [PMID:24440181]

Abstract [show]
More than 1900 different mutations in the CFTR gene have been reported. These are grouped into classes according to their effect on the synthesis and/or function of the CFTR protein. CFTR repair therapies that are mutation or mutation class specific are under development. To progress efficiently in the clinical phase of drug development, knowledge of the relative frequency of CFTR mutation classes in different populations is useful. Therefore, we describe the mutation class spectrum in 25,394 subjects with CF from 23 European countries. In 18/23 countries, 80% or more of the patients had at least one class II mutation, explained by F508del being by far the most frequent mutation. Overall 16.4% of European patients had at least one class I mutation but this varied from 3 countries with more than 30% to 4 countries with less than 10% of subjects. Overall only respectively 3.9, 3.3 and 3.0% of European subjects had at least one mutation of classes III, IV and V with again great variability: 14% of Irish patients had at least one class III mutation, 7% of Portuguese patients had at least one class IV mutation, and in 6 countries more than 5% of patients had at least one class V mutation.

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56 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations Large deletions and insertions 1078delT; 1717-1G࢐A; 3659delC; 621+1G࢐T Class II Defective protein processing G85E, F508del, I507del, R560T, N1303K Class III Defective protein regulation ('gating`) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R117H, R334W, R347P Class V Reduced amount of functioning protein 2789+5G࢐A, 3849+10KbC࢐T, A455E Unclassified All other mutations, including those unknown. Login to comment

[hide] A little CFTR goes a long way: CFTR-dependent swea... PLoS One. 2014 Feb 10;9(2):e88564. doi: 10.1371/journal.pone.0088564. eCollection 2014. Char JE, Wolfe MH, Cho HJ, Park IH, Jeong JH, Frisbee E, Dunn C, Davies Z, Milla C, Moss RB, Thomas EA, Wine JJ
A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.
PLoS One. 2014 Feb 10;9(2):e88564. doi: 10.1371/journal.pone.0088564. eCollection 2014., [PMID:24520399]

Abstract [show]
To determine if oral dosing with the CFTR-potentiator ivacaftor (VX-770, Kalydeco) improves CFTR-dependent sweating in CF subjects carrying G551D or R117H-5T mutations, we optically measured sweat secretion from 32-143 individually identified glands in each of 8 CF subjects; 6 F508del/G551D, one G551D/R117H-5T, and one I507del/R117H-5T. Two subjects were tested only (-) ivacaftor, 3 only (+) ivacaftor and 3 (+/-) ivacaftor (1-5 tests per condition). The total number of gland measurements was 852 (-) ivacaftor and 906 (+) ivacaftor. A healthy control was tested 4 times (51 glands). For each gland we measured both CFTR-independent (M-sweat) and CFTR-dependent (C-sweat); C-sweat was stimulated with a beta-adrenergic cocktail that elevated [cAMP]i while blocking muscarinic receptors. Absent ivacaftor, almost all CF glands produced M-sweat on all tests, but only 1/593 glands produced C-sweat (10 tests, 5 subjects). By contrast, 6/6 subjects (113/342 glands) produced C-sweat in the (+) ivacaftor condition, but with large inter-subject differences; 3-74% of glands responded with C/M sweat ratios 0.04%-2.57% of the average WT ratio of 0.265. Sweat volume losses cause proportionally larger underestimates of CFTR function at lower sweat rates. The losses were reduced by measuring C/M ratios in 12 glands from each subject that had the highest M-sweat rates. Remaining losses were estimated from single channel data and used to correct the C/M ratios, giving estimates of CFTR function (+) ivacaftor = 1.6%-7.7% of the WT average. These estimates are in accord with single channel data and transcript analysis, and suggest that significant clinical benefit can be produced by low levels of CFTR function.

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487 Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] Understanding how cystic fibrosis mutations disrup... Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13. Wang Y, Wrennall JA, Cai Z, Li H, Sheppard DN
Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models.
Int J Biochem Cell Biol. 2014 Jul;52:47-57. doi: 10.1016/j.biocel.2014.04.001. Epub 2014 Apr 13., [PMID:24727426]

Abstract [show]
Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients.

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1981 Interestingly, the CF mutation G1349D is the mirror image of G551D in ATP-binding site 1. Login to comment
1982 In contrast, to G551D, G1349D retains some ATP-dependence, albeit with an altered relationship between ATP concentration and channel activity (Bompadre et al., 2007). Login to comment
1983 The data suggest that G1349D disrupts CFTR function by hindering NBD dimerization (Bompadre et al., 2007). Login to comment
1984 Our own studies demonstrate that with some differences G551D and G1349D disrupt severely channel gating by decreasing the duration of channel openings and prolonging greatly the long closures that separate channel openings (Fig. 4) (Cai et al., 2006). Login to comment
1985 They also suggest that G551D-CFTR and G1349D-CFTR have distinct pharmacological profiles based on their responses to phloxine B, pyrophosphate and 2-deoxy-ATP, three agents that potentiate channel gating by different mechanisms (Cai et al., 2006). Login to comment
1986 By contrast, Yu et al. (2012) demonstrated that ivacaftor potentiated robustly both G551D-CFTR and G1349D-CFTR with similar potency, but different efficacy. Login to comment
1993 (A) Representative single-channel recordings of wild-type CFTR and the indicated CF mutants in excised inside-out membrane patches (number of active channels (N): wild-type and F508del-CFTR, N = 1; G1349D-CFTR, N = 2; G551D-CFTR, N > 4). Login to comment

[hide] Personalised medicine in cystic fibrosis is unaffo... Paediatr Respir Rev. 2014 Jun;15 Suppl 1:2-5. doi: 10.1016/j.prrv.2014.04.003. Epub 2014 Apr 13. Balfour-Lynn IM
Personalised medicine in cystic fibrosis is unaffordable.
Paediatr Respir Rev. 2014 Jun;15 Suppl 1:2-5. doi: 10.1016/j.prrv.2014.04.003. Epub 2014 Apr 13., [PMID:24832698]

Abstract [show]
Personalised medicine refers to a tailored approach to treatment of an individual based on molecular analysis of genes, proteins or metabolites, and commonly involves a companion diagnostic test. It usually applies to small subsets of patients, often with rare diseases. In cystic fibrosis (CF), the best example is the CFTR (CF transmembrane conductance regulator) potentiator, ivacaftor, relevant to the 5% of cystic fibrosis patients with the p.Gly551Asp gene mutation. However the cost of personalised medicine is too high, making it unaffordable in the long term for many healthcare systems. Society needs to find a way to make personalised medicine affordable in order to not deny life-changing treatments from patients.

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37 It is currently licensed for use only in those with the p.Gly551Asp mutation; but a further license has been recently approved in the USA for use in other rarer gating mutations (G178R, G551S, S549N, S549R, G970R, G1244E, S1251N, S1255P, or G1349D). Login to comment

[hide] New pharmacological approaches for cystic fibrosis... Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14. Bell SC, De Boeck K, Amaral MD
New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls.
Pharmacol Ther. 2015 Jan;145:19-34. doi: 10.1016/j.pharmthera.2014.06.005. Epub 2014 Jun 14., [PMID:24932877]

Abstract [show]
With the discovery of the CFTR gene in 1989, the search for therapies to improve the basic defects of cystic fibrosis (CF) commenced. Pharmacological manipulation provides the opportunity to enhance CF transmembrane conductance regulator (CFTR) protein synthesis and/or function. CFTR modulators include potentiators to improve channel gating (class III mutations), correctors to improve abnormal CFTR protein folding and trafficking (class II mutations) and stop codon mutation read-through drugs relevant for patients with premature stop codons (most class I mutations). After several successful clinical trials the potentiator, ivacaftor, is now licenced for use in adults and children (>six years), with CF bearing the class III G551D mutation and FDA licence was recently expanded to include 8 additional class III mutations. Alternative approaches for class I and class II mutations are currently being studied. Combination drug treatment with correctors and potentiators appears to be required to restore CFTR function of F508del, the most common CFTR mutation. Alternative therapies such as gene therapy and pharmacological modulation of other ion channels may be advantageous because they are mutation-class independent, however progress is less well advanced. Clinical trials for CFTR modulators have been enthusiastically embraced by patients with CF and health care providers. Whilst novel trial end-points are being evaluated allowing CFTR modulators to be efficiently tested, many challenges related to the complexity of CFTR and the biology of the epithelium still need to be overcome.

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547 Class Type of defect List of mutations attributed to this class Class I Defective protein production Nonsense mutations: G542X, R1162X, RW1282X Deletions and insertions: CFTRdele2,3; 1078delT; 1717-1G ࢐ A; 3659delC; 621+1G N T Class II Defective protein processing G85E, F508del, I507del, R560T, A561E, R1066C, N1303K Class III Defective protein regulation (gating) G178R, S549N, S549R, G551D, G551S, G970R, G1244E, S1251N, S1255P, G1349D Class IV Defective protein conductance R334W, R347P, R117H Class V Reduced amount of functioning protein 2789+5G ࢐ A, 3272-26ANG, 3849+10KbC ࢐ T, A455E Class VI Reduced cell surface stability Rescued F508del, c.120del23 Unclassified All other mutations, including those unknown a F508del-CFTR pocket (at NBD1:ICL4 interface) (Farinha et al., 2013). Login to comment

[hide] Mechanisms of CFTR functional variants that impair... PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul. LaRusch J, Jung J, General IJ, Lewis MD, Park HW, Brand RE, Gelrud A, Anderson MA, Banks PA, Conwell D, Lawrence C, Romagnuolo J, Baillie J, Alkaade S, Cote G, Gardner TB, Amann ST, Slivka A, Sandhu B, Aloe A, Kienholz ML, Yadav D, Barmada MM, Bahar I, Lee MG, Whitcomb DC
Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
PLoS Genet. 2014 Jul 17;10(7):e1004376. doi: 10.1371/journal.pgen.1004376. eCollection 2014 Jul., [PMID:25033378]

Abstract [show]
CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

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269 67 SNPs (125GtoC, 1716G.A, 1717-1G.A, 1898+1G.A, 2183AA.G, 2184delA, 2789+5G.A, 3120+1G.A, 3659delC, 3849+10kbC.T, 621+ 1G.T, 711+5G.A, A455E, D110H, D1152H, D1270N, D443Y, D579G, F1052V, F1074L, F508C, F508del, G1069R, G1244E, G1349D, G178R, G542X, G551D, G551S, I1131L/V, I148T, I336K/T, I507del, I807M, IVS8T5, K1180T, L1065P, L967S, L997F, M1V, M470V, M952I, M952T, N1303K, P67L, Q1463Q, R1070Q, R1162X, R117C, R117H, R170H, R258G, R297Q, R31C, R352Q, R553X, R668C, R74W, R75Q, S1235R, S1255P, S485R, S977F, T338I, T854T, V201M, W1282X) were multiplexed into 6 wells; 14 SNPs (S492F, S945L, R74Q, R560T, R1162L, G85E, I1027T, R334W, R347P, G576A, 711+1G.T, 1001+11C.T, P1290P, 3199del6) were ascertained separately via TaqMan Gene Expression Assays, with repeat confirmation of all positive results. Login to comment

[hide] CFTR Modulators for the Treatment of Cystic Fibros... P T. 2014 Jul;39(7):500-11. Pettit RS, Fellner C
CFTR Modulators for the Treatment of Cystic Fibrosis.
P T. 2014 Jul;39(7):500-11., [PMID:25083129]

Abstract [show]
Defects in a single gene lead to the defective proteins that cause cystic fibrosis, making the disease an ideal candidate for mutation-targeted therapy. Although ivacaftor is currently the only FDA-approved CFTR modifier, others are in development.

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36 At 48 weeks, 67% of patients in the ivacaftor group had not had a pulmonary exacerbation compared with 41% in the Table 2 Ivacaftor Clinical Trials Reference Design CFTR Mutation Population Treatment Duration Results Ramsey(2011)30 STRIVE: Randomized, double-blind, placebo-controlled G551D Age 12-53 years N = 161 FEV1 40-90% IVA 150 mg b.i.d. or PBO b.i.d. 48 wks ߦ Percent change in FEV1 from baseline to 24 wks (P < 0.001): IVA, 10.4%; PBO, -0.2% ߦ Percent change in FEV1 from baseline to 48 wks compared with PBO (P < 0.001): IVA, 10.5% ߦ Percent of patients pulmonary exacerbation-free at 48 wks: IVA, 67%; PBO, 41% ߦ Change in body weight from baseline to 48 wks: IVA, 3.1 kg; PBO, 0.4 kg ߦ Sweat chloride change from baseline to 48 wks compared with PBO (P < 0.001): IVA, -48.1 mmol/L ߦ Change in CFQ-R respiratory domain from baseline to 48 wks (P < 0.001): IVA, 5.9 pts; PBO, -2.7 pts Davies (2013)29 ENVISION: Randomized, double-blind, placebo-controlled G551D Age 6-11 years N = 52 FEV1 40-105% IVA 150 mg b.i.d. or PBO b.i.d. 48 wks ߦ Absolute change in FEV1 percentage from baseline at 48 wks compared with PBO (P < 0.001): IVA, 10% ߦ Absolute change in FEV1 percentage from baseline at 24 wks (P < 0.001): IVA, 12.6%; PBO, 0.1% ߦ Mean change in sweat chloride from baseline to 48 wks compared with PBO (P < 0.001): IVA, -54.3 mmol/L ߦ Body weight change from baseline to 48 wks compared with PBO (P < 0.001): IVA, 2.8 kg ߦ Absolute CFQ-R change from baseline to 24 wks compared with PBO (P = 0.109): IVA, 6.1 pts McKone (2013)31 PERSIST: Open-label extension G551D Age ࣙ 6 years Patients had completed 48 wks of either ENVISION or STRIVE IVA 150 mg b.i.d. 96 wks (patients received 96 wks or 144 wks of IVA depending on ENVISION or STRIVE randomization) ߦ Absolute change in percent predicted FEV1: &#b0; &#b0; STRIVE (IVA ࢐ IVA) Study start (48 wks of prior treatment): 9.4 &#b1; 8.3 &#b0; &#b0; STRIVE (IVA ࢐ IVA) 144 wks: 9.4 &#b1; 10.8 &#b0; &#b0; STRIVE (PBO ࢐ IVA) Study start: -1.2 &#b1; 7.8 &#b0; &#b0; STRIVE (PBO ࢐ IVA) 96 wks: 9.5 &#b1; 11.2 &#b0; &#b0; ENVISION (IVA ࢐ IVA) Study start (48 wks of prior treatment): 10.2 &#b1; 15.7 &#b0; &#b0; ENVISION (IVA ࢐ IVA) 144 wks: 10.3 &#b1; 12.4 &#b0; &#b0; ENVISION (PBO ࢐ IVA) Study start: -0.6 &#b1; 10.1 &#b0; &#b0; ENVISION (PBO ࢐ IVA) 96 wks: 10.5 &#b1; 11.5 ߦ Absolute change in weight (kg): &#b0; &#b0; STRIVE (IVA ࢐ IVA) Study start (48 wks of prior treatment): 3.4 &#b1; 4.9 &#b0; &#b0; STRIVE (IVA ࢐ IVA) 144 wks: 4.1 &#b1; 7.1 &#b0; &#b0; STRIVE (PBO ࢐ IVA) Study start: 0.3 &#b1; 2.2 &#b0; &#b0; STRIVE (PBO ࢐ IVA) 96 wks: 3 &#b1; 4.2 &#b0; &#b0; ENVISION (IVA ࢐ IVA) Study start (48 wks of prior treatment): 6.1 &#b1; 2.9 &#b0; &#b0; ENVISION (IVA ࢐ IVA) 144 wks: 14.8 &#b1; 5.7 &#b0; &#b0; ENVISION (PBO ࢐ IVA) Study start: 2.9 &#b1; 1.8 &#b0; &#b0; ENVISION (PBO ࢐ IVA) 96 wks: 10.1 &#b1; 4.1 ߦ Absolute change in CFQ-R respiratory domain: &#b0; &#b0; STRIVE (IVA ࢐ IVA) Study start (48 wks of prior treatment): 6.4 &#b1; 16.8 &#b0; &#b0; STRIVE (IVA ࢐ IVA) 144 wks: 6.8 &#b1; 19.6 &#b0; &#b0; STRIVE (PBO ࢐ IVA) Study start: -3.6 &#b1; 14.1 &#b0; &#b0; STRIVE (PBO ࢐ IVA) 96 wks: 9.8 &#b1; 16.2 &#b0; &#b0; ENVISION (IVA ࢐ IVA) Study start (48 wks of prior treatment): 7.4 &#b1; 17.4 &#b0; &#b0; ENVISION (IVA ࢐ IVA) 144 wks: 10.6 &#b1; 18.9 &#b0; &#b0; ENVISION (PBO ࢐ IVA) Study start: 0.8 &#b1; 18.4 &#b0; &#b0; ENVISION (PBO ࢐ IVA) 96 wks: 10.8 &#b1; 12.8 CFTR Modulators for the Treatment of Cystic Fibrosis Table 2 Ivacaftor Clinical Trials Reference Design CFTR Mutation Population Treatment Duration Results Davies (2013)32 Placebo-controlled, double-blind, crossover study G551D Age > 6 years N = 17 FEV1 > 90% LCI > 7.4 Sequence 1: PBO ࢐ WO ࢐ IVA 150 mg b.i.d. Sequence 2: IVA 150 mg b.i.d. ࢐ WO ࢐ PBO 28-day treatment and WO periods ߦ Average change in LCI from baseline compared with PBO (P < 0.0001): IVA, -2.16 (95% CI, -2.88 to -1.44) ߦ Average change in FEV1 from baseline compared with PBO (P = 0.0103): IVA, 8.67 (95% CI, 2.36 to 14.97) ߦ Average change in FEF25-75 from baseline compared with PBO (P = 0.0237): IVA, 16.56 (95% CI, 2.30 to 27.71) Barry (2013)34 Retrospective review G551D Age 20-31 in IVA group N = 21 FEV1 < 40% IVA 150 mg b.i.d. (n = 21); matched controls (n = 35) Median duration, 237 days ߦ Absolute FEV1 change from baseline (P = 0.0075): IVA, 0.125 L; CON, 0.01 L ߦ Percent predicted FEV1 change from baseline (P = 0.0092): IVA, 12.7%, CON, 2.2% ߦ Median weight increase from baseline: IVA, 1.8 kg; CON, 0.1 kg ߦ Median inpatient days per year decreased from 23 days to 0 days in the IVA group (P = 0.001) ߦ Median total intravenous antibiotic days per year decreased from 74 days to 38 days in the IVA group (P = 0.002) De Boeck (2013)37 KONNECTION: Randomized, double-blind, crossover, placebo-controlled Non-G551D gating mutations G178R, G551S, S549N, S549R, G970R, G1244E, S1251N, S1255P, G1349D Age ࣙ 6 years N = 39 FEV1 ࣙ 40% Treatment sequence 1: IVA 150 mg b.i.d. ࢐ WO ࢐ PBO ࢐ open-label Treatment sequence 2: PBO ࢐ WO ࢐ IVA 150 mg b.i.d. ࢐ open-label 8 wks of IVA or PBO; 4-8 wks WO period; 16 wks open label ߦ Absolute change from baseline percent predicted FEV1 (P < 0.0001): IVA, 7.49%; PBO, -3.19% ߦ Absolute change from baseline BMI (P < 0.0001): IVA, 0.68; PBO, 0.02 ߦ Absolute change from baseline in CFQ-R respiratory domain (P = 0.0004): IVA, 8.94 pts; PBO, -0.67 pts ߦ Absolute change from baseline in sweat chloride (mmol/L): IVA, -52.28; PBO, -3.11 Flume (2011)35 Randomized, double-blind, placebo-controlled, parallel group with open-label extension Homozygous F508del Age ࣙ 12 years Part 1: N = 140 Part 2: N = 33 42 patients were eligible for part 2 if change in FEV1 ࣙ 10% or sweat chloride decreased by at least 15 mmol/L at day 15 and week 8 Part 1: IVA 150 mg b.i.d. or PBO 16 wks Part 2: Open label IVA 150 mg b.i.d. 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56 These promising results led to an FDA label expansion to include CF patients with the following eight mutations in addition to G551D: G178R, S549R, S549N, G551S, G1244E, S1251N, S1255P, and G1349D.38 Clinical Considerations Ivacaftor was well tolerated in clinical trials. Login to comment

[hide] A cocktail drug therapy for patients with cystic f... J Cyst Fibros. 2014 Sep;13(5):489-90. doi: 10.1016/j.jcf.2014.07.002. Epub 2014 Jul 24. Chen JH
A cocktail drug therapy for patients with cystic fibrosis?
J Cyst Fibros. 2014 Sep;13(5):489-90. doi: 10.1016/j.jcf.2014.07.002. Epub 2014 Jul 24., [PMID:25088968]

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6 More recently, VX-770 has been approved by the FDA (NDA 203188, www.fda.gov) and recommended by the EMA (EMA/CHMP/365663/2014) for use with an additional eight CF gating (class III) mutations (G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D), although, including G551D, these mutations still just occur in ~5% of CF patients worldwide. Login to comment

[hide] A single amino acid substitution in CFTR converts ... J Gen Physiol. 2014 Oct;144(4):311-20. doi: 10.1085/jgp.201411247. Epub 2014 Sep 15. Lin WY, Jih KY, Hwang TC
A single amino acid substitution in CFTR converts ATP to an inhibitory ligand.
J Gen Physiol. 2014 Oct;144(4):311-20. doi: 10.1085/jgp.201411247. Epub 2014 Sep 15., [PMID:25225552]

Abstract [show]
Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Furthermore, decreasing [ATP] from 2 mM to 20 microM resulted in a paradoxical increase in G551D-CFTR current. These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the gamma-phosphate of ATP accounts for the observed mutational effects. Understanding the molecular mechanism of this gating defect lays a foundation for rational drug design for the treatment of CF.

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228 G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] Full-open and closed CFTR channels, with lateral t... Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7. Mornon JP, Hoffmann B, Jonic S, Lehn P, Callebaut I
Full-open and closed CFTR channels, with lateral tunnels from the cytoplasm and an alternative position of the F508 region, as revealed by molecular dynamics.
Cell Mol Life Sci. 2015 Apr;72(7):1377-403. doi: 10.1007/s00018-014-1749-2. Epub 2014 Oct 7., [PMID:25287046]

Abstract [show]
In absence of experimental 3D structures, several homology models, based on ABC exporter 3D structures, have provided significant insights into the molecular mechanisms underlying the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, a chloride channel whose defects are associated with cystic fibrosis (CF). Until now, these models, however, did not furnished much insights into the continuous way that ions could follow from the cytosol to the extracellular milieu in the open form of the channel. Here, we have built a refined model of CFTR, based on the outward-facing Sav1866 experimental 3D structure and integrating the evolutionary and structural information available today. Molecular dynamics simulations revealed significant conformational changes, resulting in a full-open channel, accessible from the cytosol through lateral tunnels displayed in the long intracellular loops (ICLs). At the same time, the region of nucleotide-binding domain 1 in contact with one of the ICLs and carrying amino acid F508, the deletion of which is the most common CF-causing mutation, was found to adopt an alternative but stable position. Then, in a second step, this first stable full-open conformation evolved toward another stable state, in which only a limited displacement of the upper part of the transmembrane helices leads to a closure of the channel, in a conformation very close to that adopted by the Atm1 ABC exporter, in an inward-facing conformation. These models, supported by experimental data, provide significant new insights into the CFTR structure-function relationships and into the possible impact of CF-causing mutations.

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360 The effects of remaining mutations listed in CFTR2 database can also be well understood in light of our structural data: (1) A455E has indeed no room to be well adapted, (2) G1244E (mentioned above) also occurs in a well conserved position (position 23 in Online Resource 2), (3) L467P might disturb the helix in which it is included, (4) G1349D is in the non-canonical ATP-binding site and, alike its corresponding G551D, has no room to be well adapted in presence of ATP, and finally (5) N1303K might disturb the large Q-loop of the NBD2 a-helical subdomain (position 42 in Online Resource 1 and Online Resource 2). Login to comment

[hide] Analysis of cystic fibrosis gene mutations in chil... J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339. Dell'Edera D, Benedetto M, Gadaleta G, Carone D, Salvatore D, Angione A, Gallo M, Milo M, Pisaturo ML, Di Pierro G, Mazzone E, Epifania AA
Analysis of cystic fibrosis gene mutations in children with cystic fibrosis and in 964 infertile couples within the region of Basilicata, Italy: a research study.
J Med Case Rep. 2014 Oct 10;8:339. doi: 10.1186/1752-1947-8-339., [PMID:25304080]

Abstract [show]
INTRODUCTION: Cystic fibrosis is the most common autosomal recessive genetic disease in the Caucasian population. Extending knowledge about the molecular pathology on the one hand allows better delineation of the mutations in the CFTR gene and the other to dramatically increase the predictive power of molecular testing. METHODS: This study reports the results of a molecular screening of cystic fibrosis using DNA samples of patients enrolled from January 2009 to December 2013. Patients were referred to our laboratory for cystic fibrosis screening for infertile couples. In addition, we identified the gene mutations present in 76 patients affected by cystic fibrosis in the pediatric population of Basilicata. RESULTS: In the 964 infertile couples examined, 132 subjects (69 women and 63 men) resulted heterozygous for one of the CFTR mutations, with a recurrence of carriers of 6.85%. The recurrence of carriers in infertile couples is significantly higher from the hypothetical value of the general population (4%). CONCLUSIONS: This study shows that in the Basilicata region of Italy the CFTR phenotype is caused by a small number of mutations. Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for screened couples is superimposable with respect to the general population.

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59 As mentioned before, molecular screening Table 2 Comparison between the results obtained in this study and those obtained in a previous study Castaldo et al. [14] Mutations observed in the present study F508del 55.8% (29) 48.62% (141) N1303K 3.8% (2) 9.31% (27) G542X 3.8% (2) 8.96% (26) W1282X 3.8% (2) 1.03% (3) 2183AA>G 5.8% (3) 2.76% (8) R1162X 0 0 1717-1G>A 1.9% (1) 0 T338I 0 0 R347P 0 0.69% (2) 711+5G>A 0 0 852del22 5.8% (3) 1.03% (3) 4382delA 0 0.69% (2) 1259insA 0 0.34% (1) 4016insT 0 0.34% (1) R553X 0 0.34% (1) R1158X 0 0 L1077P 0 1.03% (3) I502T 0 0 3849+10kbC>T 1.9% (1) 0.34% (1) D579G 0 0.69% (2) G1244E 3.8% (2) 0 G1349D 0 0.34% (1) 2789+5G>A 0 1.03% (3) 711+1G>T 0 0 L1065P 0 0 2522insC 0 0 E585X 0 0 G85E 0 0 G178R 0 0 D1152H 0 3.10% (9) I148T-3195del6 0 0 I148T (alone) 0 4.48% (13) R334W 0 0 DI507 0 0.69% (2) I1005R 0 0 3272-26A>G 0 0 2711delT 0 0 L558S 1.9% (1) 0.34% (1) W1063X 0 0 D110H 0 0 S549R (A>C) 1.9% (1) 0.69% (2) 2184insA 0 0 3131del22 0 0 Table 2 Comparison between the results obtained in this study and those obtained in a previous study (Continued) R709N 0 0 A349V 0 0 4015insA 0 0 Y849X 1.9% (1) 0.34% (1) G551D 0 1.03% (3) 621+3A>G 0 0.34% (1) E831X 0 0 I507del 0 0.69% (2) IVS8 TG12/t5 0 1.03% (3) H139R (A->G) 0 0.34% (1) 1248+1G>A 0 0.34% (1) R74W;V201M;D1270N 0 0.69% (2) S1455X 0 0.34% (1) dele 2,3 (21kb) 0 0.34% (1) 991del5 0 0.34% (1) UNKNOWN 7 %(4) 4.83% (14) F508C 0 0.69% (2) TOTAL 52 290 of CF is highly recommended in the USA by the National Institutes of Health Consensus Development Conference Statement on genetic testing for cystic fibrosis [17]. Login to comment
79 The test has a sensitivity and a specificity of more than Table 3 List of 60 mutations in the cystic fibrosis transmembrane regulator gene (specificity 100%) F508del I507del F508C 621+1G>T D110H E585X G1349D I502T 1706del17 1677delTA R117H H139R 1898+1G>A 4015delA G542X 1717-1G>A Q552X 852del22 G178R 1898+3A>G G551D S549R(A>C) 2183AA>G T338I 991del5 1898+5G>T N1303K 4016insT 3849+10kb C>T R347P R334W 2184insA G85E 711+5G>A 711+1G>T 1259insA R347H 2522insC 2789+5G>A W1282X G1244E R1066H R352Q 3120+1G>A I148T 3199del6 S912X R1158X 1717-8G>A R1066C R1162X 4382delA D1152H L1077P D579G 3272-26A>G L1065P R553X PoliT: 5T, 7T, 9T 1874insT 3659delC 99%. Login to comment

[hide] Localizing a gate in CFTR. Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9. Gao X, Hwang TC
Localizing a gate in CFTR.
Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):2461-6. doi: 10.1073/pnas.1420676112. Epub 2015 Feb 9., [PMID:25675504]

Abstract [show]
Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR's gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2](-), we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338-341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2](-) in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2](-) in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2](-) when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2](-) and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.

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4 However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2]- in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2]- when the open probability is markedly reduced by introducing a second mutation, G1349D. Login to comment
96 Our previous studies demonstrated that a disease-associated mutation G1349D could decrease the Po of CFTR by ~10-fold (34) without affecting trafficking of the channel (34, 35); we thus engineered this mutation into R334C, K335C, F337C, and T338C backgrounds. Login to comment
97 As shown in Fig. S5, indeed, introducing the G1349D mutation into T338C-CFTR lowered the Po and decreased the reaction rate by ~10-fold, which can be interpreted as a limited accessibility of the side chain of 338C in the closed state. Login to comment
98 However, the reaction rate of extracellularly applied [Au(CN)2]- for the 337C/G1349D mutant is slightly but noticeably faster than that of the 337C mutant (Fig. S5B), suggesting that the 337C side chain is better exposed in the closed state. Login to comment
102 Similar experiments were performed with the double mutant R334C/G1349D (Fig. 3B). Login to comment
103 Fitting the current decays upon addition of [Au(CN)2]- yielded the reaction rates of 403 &#b1; 20 /M/s (n = 7) and 537 &#b1; 56 /M/s (n = 6) for R334C and R334C/G1349D, respectively. Login to comment
104 Although the Po of R334C-CFTR cannot be assessed due to a greatly reduced single-channel amplitude, by comparing macroscopic current amplitudes in a large number of patches (Fig. 3E), we verified a more than 10-fold decrease of Po by introducing the G1349D mutation. Login to comment
110 However, after G1349D was introduced into K335C-CFTR to lower its Po, 50 bc;M [Au(CN)2]- could readily react with 335C with a reaction rate of 1,809 &#b1; 201 /M/s (n = 5) (Fig. 3D). Login to comment
143 (B) [Au(CN)2]- reacted with R334C/G1349D at a faster rate than that with R334C. Login to comment
149 (D) Reaction of K335C/G1349D-CFTR with [Au(CN)2]- . Login to comment
153 (E) Comparisons of the mean current amplitude between R334C-CFTR and R334C/G1349D-CFTR and between K335C-CFTR and K335C/G1349D-CFTR in excised inside-out patches. Login to comment
154 Note that a ~10-fold difference in the mean current amplitude-hence a ~10-fold change in Po-was seen with the introduction of the G1349D mutation, as shown previously for WT-CFTR (see Results for details). Login to comment
192 [Au(CN)2]- , forskolin, with G1349D, /M/s Outside R334C 189 &#b1; 39 - 403 &#b1; 20 537 &#b1; 56 K335C - - 56 &#b1; 9 1,809 &#b1; 201 F337C 437 &#b1; 49 - 20 &#b1; 3 32 &#b1; 6 T338C 752 &#b1; 59 - 1,135 &#b1; 166 118 &#b1; 18 Inside I344C 32 &#b1; 5 37 &#b1; 4 - - N1148C 437 &#b1; 66 2,089 &#b1; 130 - - Residues located extracellularly (extra.) Login to comment
318 Bompadre SG, Sohma Y, Li M, Hwang TC (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment

[hide] [CFTR gene sequencing in a group of Chilean patien... Rev Chil Pediatr. 2014 Jul;85(4):448-54. doi: 10.4067/S0370-41062014000400007. Lay-Son R G, Vasquez D M, Puga Y A, Manque M P, Repetto L G
[CFTR gene sequencing in a group of Chilean patients with cystic fibrosis].
Rev Chil Pediatr. 2014 Jul;85(4):448-54. doi: 10.4067/S0370-41062014000400007., [PMID:25697318]

Abstract [show]
INTRODUCTION: Cystic fibrosis (CF) is an autosomal recessive genetic disorder caused by mutations of the CFTR gene, in which over 1,900 different mutations have been identified. In Chile, the diagnosis panel with the 36 most common mutations detects approximately 50% of all alleles, while for Caucasians, it is nearly 90%. The objective of this study is to expand the capacity of mutational screening in Chilean patients and look for recurrent mutations at the national level. METHOD: The detection of unknown pathogenic alleles was assessed by CFTR gene sequencing in a selected group of patients from the National Cystic Fibrosis Foundation (NCFF). 39 patients, who met the CF diagnostic criteria and had only one allele identified according to the mutational panel, were studied. Massive sequencing was performed throughout the investigation and the main CFTR databases were used for analysis. RESULTS: The second pathogenic allele was identified in 16 of 39 patients of this study (41%), finding eleven different mutations that had not been reported in our population. We believe that the reason is that one of the variants had not been previously described. CONCLUSIONS: Mutations that had been described mainly in Hispanic and/or Mediterranean populations were identified. We found a variation that had not been previously reported, but not enough recurrent mutations that could explain the low rate of detection were found. Knowledge about mutations can provide appropriate genetic counseling and will be critical to evaluate the potential use of new targeted therapies for treating them.

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58 Mutaciones detectadas por secuenciaci&#f3;n masiva en cohorte de 39 pacientes chilenos con FQ portadores de un alelo desconocido Mutaci&#f3;n detectada (nomenclatura actual*) n de alelos Reporte en pacientes con FQ (no de alelos) Efecto Denominaci&#f3;n antigua c.1330_1331delAT 3 Argentina (1)a Prote&#ed;na truncada por generaci&#f3;n de cod&#f3;n de t&#e9;rmino 1460delAT c.314T>A 2 Francia (1)a Cambio de amino&#e1;cido Isoleucina por Asparagina I105N c.4046G>A 2 Italia (7)b,c , EEUU (1)d Cambio de amino&#e1;cido Glicina por Aspartato G1349D c.148T>C 2 Espa&#f1;a (2)e Cambio de amino&#e1;cido Serina por Prolina S50P c.695T>A 1 Espa&#f1;a (14)e,f , EEUU (hispanos) (5)g,h Francia (2)a , Brasil (1)i Cambio de amino&#e1;cido Valina por Aspartato V232D c.3266G>A 1 Espa&#f1;a (5)e , Brasil (2)i,j , EEUU (hispanos) (2)g , Argentina (1)k , Israel (1)l Prote&#ed;na truncada por generaci&#f3;n de cod&#f3;n de t&#e9;rmino W1089X c.1647T>G 1 Emiratos &#c1;rabes Unidos (> 30)m,n , Colombia (4)o , Israel (4)p , Argelia (2)p , Marruecos (2)q , Reino Unido (2)p , Portugal (1)p , Espa&#f1;a (1)p , Francia (1)p , Italia (1)p , Brasil (1)q , Argentina (1)q Cambio de amino&#e1;cido Serina por Arginina S549R(T- >G) c.308G>A 1 No descrita previamente Cambio de amino&#e1;cido Glicina por Glutamato G103E c.1680-1G>A 1 Espa&#f1;a (1)r Alteraci&#f3;n en splicing 1812-1G->A c.1679+1G>C 1 Francia (2)s Macedonia (1)s , Alteraci&#f3;n en splicing 1811+1G->C c.490-2A>G 1 Argentina (1)t Alteraci&#f3;n en splicing 622-2A->G FQ: Fibrosis qu&#ed;stica. Login to comment

[hide] A rapid molecular approach for chromosomal phasing... PLoS One. 2015 Mar 4;10(3):e0118270. doi: 10.1371/journal.pone.0118270. eCollection 2015. Regan JF, Kamitaki N, Legler T, Cooper S, Klitgord N, Karlin-Neumann G, Wong C, Hodges S, Koehler R, Tzonev S, McCarroll SA
A rapid molecular approach for chromosomal phasing.
PLoS One. 2015 Mar 4;10(3):e0118270. doi: 10.1371/journal.pone.0118270. eCollection 2015., [PMID:25739099]

Abstract [show]
Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

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57 The cystic fibrosis cell lines derived from Epstein-Barr virus transformed B-lymphocytes included: GM11286 and GM11274, which were determined to have c.1652G>A (p.Gly551Asp) and c.1521_1523delCTT (p.Phe508del) variants [22]; GM11279, which was determined to have 129G>C (promoter), c.350G>A (p.Arg117His), and c.1521_1523delCTT (p.Phe508del) variants [23]; GM11472, which was characterized to have c.1210-12T[7], c.1210-12T[9], c.3909C>G (p. Asn1303Lys), and c.4046G>A (p.Gly1349Asp) variants [24,25] (c.4046G>A is also referred to as c.4178G>A in some dbSNP databases); and GM13591, which was characterized to have c.350G>A (p.Arg117His), c.1210-12T[5], c.1210-12T[9], and c.1521_1523delCTT (p.Phe508del) variants [26]. Login to comment
180 Variant 129G/C R117H 5T 7T 9T ƊF508 G551D N1303K G1349D Effect on cDNA promoter 350G>A intron intron intron 1521_1523 delCTT 1652G>A 3909C>G 4046G>A (4178G>A) GM11286 Hap 1 Hap 2 GM03465 Hap 1 Hap 2 GM11274 Hap 1 Hap 2 GM11279 Hap 1 Hap 1 Hap 1 Hap 2 Hap 2 GM11472 Hap 1 Hap 2 Hap 2 Hap 1 GM13591 Hap 1 Hap 1 Hap 2 Hap 2 Key: Hap 1 = Haplotype 1, Hap 2 = Haplotype 2 doi:10.1371/journal.pone.0118270.t001 Fig 4. Login to comment

[hide] Functional reconstitution and channel activity mea... J Vis Exp. 2015 Mar 9;(97). doi: 10.3791/52427. Eckford PD, Li C, Bear CE
Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein.
J Vis Exp. 2015 Mar 9;(97). doi: 10.3791/52427., [PMID:25867140]

Abstract [show]
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

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30 While the correctors VX-809 and VX-661 (are not yet approved for use in patients, the potentiator Kalydeco (ivacaftor; VX-770) is being used at 150 mg every 12 hr in CF patients >6 years with at least one G551D-CFTR mutation, and more recently for patients with one of G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D. Login to comment

[hide] A Genotypic-Oriented View of CFTR Genetics Highlig... Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229. Lucarelli M, Bruno SM, Pierandrei S, Ferraguti G, Stamato A, Narzi F, Amato A, Cimino G, Bertasi S, Quattrucci S, Strom R
A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis.
Mol Med. 2015 Apr 21;21:257-75. doi: 10.2119/molmed.2014.00229., [PMID:25910067]

Abstract [show]
Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype-phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype-phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.

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390 L1077P c.3230T>C CF-PI CF-causing p.Leu1077Pro Y1092X(C>A) c.3276C>A CF-PI CF-causing p.Tyr1092* M1137V c.3409A>G CFTR-RD nd p.Met1137Val D1152H c.3454G>C CF-PI,CF-PS,CFTR-RD varying clinical consequence p.Asp1152His R1162X c.3484C>T CF-PI CF-causing p.Arg1162* D1168G c.3503A>G CFTR-RD nd p.Asp1168Gly 3667ins4 c.3535_3536insTCAA CF-PI CF-causing p.Thr1179IlefsX17 S1206X c.3617C>A uncertain: CF-PI and/or CF-PS nd p.Ser1206* I1234V c.3700A>G CF-PI,CF-PS CF-causing p.Ile1234Val S1235R c.3705T>G CFTR-RD non CF-causing p.Ser1235Arg 3849+10kbC>T c.3717+12191C>T CF-PI,CF-PS CF-causing V1240G c.3719T>G CFTR-RD nd p.Val1240Gly G1244R c.3730G>A uncertain: CF-PI and/or CF-PS nd p.Gly1244Arg G1244E c.3731G>A CF-PI,CF-PS CF-causing p.Gly1244Glu G1247R(G>C) c.3739G>C CF-PS nd p.Gly1247Arg W1282X c.3846G>A CF-PI CF-causing p.Trp1282* Q1291R c.3872A>G CF-PI,CF-PS,CFTR-RD nd p.Gln1291Arg 4016insT c.3884_3885insT CF-PI CF-causing p.Ser1297PhefsX5 4040delA c.3908delA CF-PI nd p.Asn1303ThrfsX25 N1303K c.3909C>G CF-PI CF-causing p.Asn1303Lys ex22-24del c.3964-3890_4443+3143del9454ins5 CF-PI nd ex22,23del c.3964-78_4242+577del1532 CF-PI CF-causing 4168delCTAAGCC c.4036_4042del CF-PI nd p.Leu1346MetfsX6 G1349D c.4046G>A CF-PI CF-causing p.Gly1349Asp H1375P c.4124A>C uncertain: CF-PI and/or CF-PS nd p.His1375Pro S1455X c.4364C>G CF-PS,CFTR-RD nd p.Ser1455* Q1476X c.4426C>T CFTR-RD nd p.Gln1476* nd,Not determined.According to the three rules described (see Materials and Methods),each mutated allele was classified according to its clinical outcome.It was impossible to univocally assign 16 of the 125 different mutated alleles to one or more macrocategories.A comparison with the CFTR2 project (11) (http://www.cftr2.org) is shown.The alleles are ordered according to their nucleotidic position. Login to comment

[hide] Translating the genetics of cystic fibrosis to per... Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008. Corvol H, Thompson KE, Tabary O, le Rouzic P, Guillot L
Translating the genetics of cystic fibrosis to personalized medicine.
Transl Res. 2015 Apr 15. pii: S1931-5244(15)00131-0. doi: 10.1016/j.trsl.2015.04.008., [PMID:25940043]

Abstract [show]
Cystic fibrosis (CF) is the most common life-threatening recessive genetic disease in the Caucasian population. This multiorgan disease is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a chloride channel recognized as regulating several apical ion channels. The gene mutations result either in the lack of the protein at the apical surface or in an improperly functioning protein. Morbidity and mortality because of the mutation of CFTR are mainly attributable to lung disease resulting from chronic infection and inflammation. Since its discovery as the causative gene in 1989, much progress has been achieved not only in clinical genetics but also in basic science studies. Recently, combinations of these efforts have been successfully translated into development and availability for patients of new therapies targeting specific CFTR mutations to correct the CFTR at the protein level. Current technologies such as next gene sequencing and novel genomic editing tools may offer new strategies to identify new CFTR variants and modifier genes, and to correct CFTR to pursue personalized medicine, which is already developed in some patient subsets. Personalized medicine or P4 medicine ("personalized," "predictive," "preventive," and "participatory") is currently booming for CF. The various current and future challenges of personalized medicine as they apply to the issues faced in CF are discussed in this review.

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155 Furthermore, Kalydeco has been tested in patients carrying other class III mutations, or targeted class IVand V mutations (sharing functional similarities with the class III).58 The new trials led to an extension of the FDA and European Medical Agency approval to 8 additional gating mutations: p.Gly178Arg (p.G178R), p.Ser549Asn (p.S549N), p.Ser549Arg (p.S549R), p.Gly551Ser (p.G551S), p.Gly1244Glu (p.G1244E), p.Ser1251Asn (p.S1251N), p.Ser1255Pro (pS1255P), and p.Gly1349Asp (p.G1349D).59 Recently, ivacaftor has also been shown to benefit patients carrying the c.350G . Login to comment

[hide] [Challenges of personalized medicine for cystic fi... Arch Pediatr. 2015 Jul;22(7):778-86. doi: 10.1016/j.arcped.2015.04.015. Epub 2015 May 26. Corvol H, Taytard J, Tabary O, Le Rouzic P, Guillot L, Clement A
[Challenges of personalized medicine for cystic fibrosis].
Arch Pediatr. 2015 Jul;22(7):778-86. doi: 10.1016/j.arcped.2015.04.015. Epub 2015 May 26., [PMID:26021452]

Abstract [show]
Personalized medicine, or P4 medicine for "Personalized", "Predictive", "Preventive" and "Participatory", is currently booming for cystic fibrosis, with the development of therapies targeting specific CFTR mutations. The various challenges of personalized medicine applied to cystic fibrosis issues are discussed in this paper.

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135 Compte tenu de l`efficacite &#b4; de KalydecoW chez ces patients, le laboratoire VertexW a ensuite teste &#b4;, puis de &#b4;montre &#b4; son efficacite &#b4; chez des patients porteurs d`autres mutations de classe III, ce qui a permis cette anne &#b4;e une extension d`autorisation de mise sur le marche &#b4; (AMM) pour 8 mutations supple &#b4;mentaires : G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N et S549R [37]. Login to comment

[hide] Targeting ion channels in cystic fibrosis. J Cyst Fibros. 2015 Sep;14(5):561-70. doi: 10.1016/j.jcf.2015.06.002. Epub 2015 Jun 23. Mall MA, Galietta LJ
Targeting ion channels in cystic fibrosis.
J Cyst Fibros. 2015 Sep;14(5):561-70. doi: 10.1016/j.jcf.2015.06.002. Epub 2015 Jun 23., [PMID:26115565]

Abstract [show]
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause a characteristic defect in epithelial ion transport that plays a central role in the pathogenesis of cystic fibrosis (CF). Hence, pharmacological correction of this ion transport defect by targeting of mutant CFTR, or alternative ion channels that may compensate for CFTR dysfunction, has long been considered as an attractive approach to a causal therapy of this life-limiting disease. The recent introduction of the CFTR potentiator ivacaftor into the therapy of a subgroup of patients with specific CFTR mutations was a major milestone and enormous stimulus for seeking effective ion transport modulators for all patients with CF. In this review, we discuss recent breakthroughs and setbacks with CFTR modulators designed to rescue mutant CFTR including the common mutation F508del. Further, we examine the alternative chloride channels TMEM16A and SLC26A9, as well as the epithelial sodium channel ENaC as alternative targets in CF lung disease, which remains the major cause of morbidity and mortality in patients with CF. Finally, we will focus on the hurdles that still need to be overcome to make effective ion transport modulation therapies available for all patients with CF irrespective of their CFTR genotype.

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604 When tested in clinical trials, the potentiator ivacaftor (also known as VX-770) showed a marked clinical benefit, with substantial improvement of lung function, reduction of pulmonary exacerbations, and increase in body weight in CF patients with G551D and 8 additional Class III mutations (G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D) [32-35]. Login to comment

[hide] Hallmarks of therapeutic management of the cystic ... J Cyst Fibros. 2015 Nov;14(6):687-99. doi: 10.1016/j.jcf.2015.09.006. Epub 2015 Oct 29. Amaral MD, Balch WE
Hallmarks of therapeutic management of the cystic fibrosis functional landscape.
J Cyst Fibros. 2015 Nov;14(6):687-99. doi: 10.1016/j.jcf.2015.09.006. Epub 2015 Oct 29., [PMID:26526359]

Abstract [show]
The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein does not operate in isolation, rather in a dynamic network of interacting components that impact its synthesis, folding, stability, intracellular location and function, referred to herein as the 'CFTR Functional Landscape (CFFL)'. For the prominent F508del mutation, many of these interactors are deeply connected to a protein fold management system, the proteostasis network (PN). However, CF encompasses an additional 2000 CFTR variants distributed along its entire coding sequence (referred to as CFTR2), and each variant contributes a differential liability to PN management of CFTR and to a protein 'social network' (SN) that directs the probability of the (patho)physiologic events that impact ion transport in each cell, tissue and patient in health and disease. Recognition of the importance of the PN and SN in driving the unique patient CFFL leading to disease highlights the importance of precision medicine in therapeutic management of disease progression. We take the view herein that it is not CFTR, rather the PN/SN, and their impact on the CFFL, that are the key physiologic forces driving onset and clinical progression of CF. We posit that a deep understanding of each patients PN/SN gained by merging genomic, proteomic (mass spectrometry (MS)), and high-content microscopy (HCM) technologies in the context of novel network learning algorithms will lead to a paradigm shift in CF clinical management. This should allow for generation of new classes of patient specific PN/SN directed therapeutics for personalized management of the CFFL in the clinic.

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656 The FDA approval of Ivacaftor for multiple G551D like phenotypic variants including G178R, S549N, S549R, G551S, G1244E, S1251N, S1255P and G1349D [159,160] found at the cell surface with gating defects [161,162], and the FDA-approval of a combination of Lumacaftor and Ivacaftor for treatment of F508del [156] are examples of successful application of these technologies. Login to comment