ABCMdb

ABCC7 p.Gly1349Asp

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III (gating defect) <a href="https://www.ncbi.nlm.nih.gov/pubmed/26823392">Veit et al.</a>
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Publications

PMID: 10021451 [PubMed] Zeitlin PL et al: "Novel pharmacologic therapies for cystic fibrosis."

No. Sentence Comment

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

PMID: 10720936 [PubMed] Zeitlin PL et al: "Pharmacologic restoration of delta F508 CFTR-mediated chloride current."

No. Sentence Comment

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

PMID: 10777364 [PubMed] Wang J et al: "A novel mutation in the CFTR gene correlates with severe clinical phenotype in seven Hispanic patients."

No. Sentence Comment

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

PMID: 10777368 [PubMed] Rivard SR et al: "Correlation between mutations and age in cystic fibrosis in a French Canadian population."

No. Sentence Comment

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

PMID: 11100963 [PubMed] Choo-Kang LR et al: "Type I, II, III, IV, and V cystic fibrosis transmembrane conductance regulator defects and opportunities for therapy."

No. Sentence Comment

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

PMID: 11242048 [PubMed] Choi JY et al: "Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis."

No. Sentence Comment

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

PMID: 12124743 [PubMed] Salvatore F et al: "Genotype-phenotype correlation in cystic fibrosis: the role of modifier genes."

No. Sentence Comment

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

PMID: 12457238 [PubMed] Ando-Akatsuka Y et al: "Down-regulation of volume-sensitive Cl- channels by CFTR is mediated by the second nucleotide-binding domain."

No. Sentence Comment

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

PMID: 14662004 [PubMed] Zeitlin PL et al: "Emerging drug treatments for cystic fibrosis."

No. Sentence Comment

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

PMID: 15638824 [PubMed] Castaldo G et al: "Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italian population."

No. Sentence Comment

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

PMID: 15705292 [PubMed] Claustres M et al: "Molecular pathology of the CFTR locus in male infertility."

No. Sentence Comment

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

PMID: 15719171 [PubMed] Moran O et al: "Binding site of activators of the cystic fibrosis transmembrane conductance regulator in the nucleotide binding domains."

No. Sentence Comment

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

PMID: 15722457 [PubMed] Pedemonte N et al: "Phenylglycine and sulfonamide correctors of defective delta F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating."

No. Sentence Comment

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

PMID: 16311240 [PubMed] Cai Z et al: "Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel."

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

PMID: 16472140 [PubMed] Becq F et al: "On the discovery and development of CFTR chloride channel activators."

No. Sentence Comment

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

PMID: 16604470 [PubMed] Melin P et al: "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."

No. Sentence Comment

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

PMID: 16787252 [PubMed] Verkman AS et al: "CFTR chloride channel drug discovery--inhibitors as antidiarrheals and activators for therapy of cystic fibrosis."

No. Sentence Comment

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

PMID: 17244607 [PubMed] Zegarra-Moran O et al: "Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition."

No. Sentence Comment

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

PMID: 17353351 [PubMed] Bompadre SG et al: "G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects."

No. Sentence Comment

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

PMID: 17452495 [PubMed] Pedemonte N et al: "Structure-activity relationship of 1,4-dihydropyridines as potentiators of the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

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

PMID: 17578899 [PubMed] Routaboul C et al: "Discovery of alpha-aminoazaheterocycle-methylglyoxal adducts as a new class of high-affinity inhibitors of cystic fibrosis transmembrane conductance regulator chloride channels."

No. Sentence Comment

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

PMID: 17700963 [PubMed] Bompadre SG et al: "Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis."

No. Sentence Comment

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

PMID: 17890437 [PubMed] Montgomery J et al: "Scanning the cystic fibrosis transmembrane conductance regulator gene using high-resolution DNA melting analysis."

No. Sentence Comment

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

PMID: 18167357 [PubMed] Bompadre SG et al: "Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog."

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

PMID: 18178635 [PubMed] Stanke F et al: "Diversity of the basic defect of homozygous CFTR mutation genotypes in humans."

No. Sentence Comment

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

PMID: 18193900 [PubMed] Cheung JC et al: "Misfolding of the cystic fibrosis transmembrane conductance regulator and disease."

No. Sentence Comment

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

PMID: 18230692 [PubMed] Norez C et al: "Proteasome-dependent pharmacological rescue of cystic fibrosis transmembrane conductance regulator revealed by mutation of glycine 622."

No. Sentence Comment

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

PMID: 18251718 [PubMed] Landry Y et al: "Drugs and their molecular targets: an updated overview."

No. Sentence Comment

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

PMID: 18366345 [PubMed] Caci E et al: "Evidence for direct CFTR inhibition by CFTR(inh)-172 based on Arg347 mutagenesis."

No. Sentence Comment

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

PMID: 18391167 [PubMed] Chen TY et al: "CLC-0 and CFTR: chloride channels evolved from transporters."

No. Sentence Comment

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

PMID: 19114635 [PubMed] Wang X et al: "Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel."

No. Sentence Comment

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

PMID: 19332488 [PubMed] Hwang TC et al: "Gating of the CFTR Cl- channel by ATP-driven nucleotide-binding domain dimerisation."

No. Sentence Comment

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

PMID: 19332621 [PubMed] Tsai MF et al: "State-dependent modulation of CFTR gating by pyrophosphate."

No. Sentence Comment

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

PMID: 19491324 [PubMed] Caputo A et al: "Mutation-specific potency and efficacy of cystic fibrosis transmembrane conductance regulator chloride channel potentiators."

No. Sentence Comment

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

PMID: 19837660 [PubMed] Chen JH et al: "Direct sensing of intracellular pH by the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel."

No. Sentence Comment

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

PMID: 19880323 [PubMed] Cateni F et al: "Synthesis of 4-thiophen-2'-yl-1,4-dihydropyridines as potentiators of the CFTR chloride channel."

No. Sentence Comment

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

PMID: 19903491 [PubMed] Kreindler JL et al: "Cystic fibrosis: exploiting its genetic basis in the hunt for new therapies."

No. Sentence Comment

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

PMID: 19910675 [PubMed] Treharne KJ et al: "Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR."

No. Sentence Comment

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

PMID: 20053923 [PubMed] Pedemonte N et al: "Influence of cell background on pharmacological rescue of mutant CFTR."

No. Sentence Comment

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

PMID: 20133716 [PubMed] Wang W et al: "ATP-independent CFTR channel gating and allosteric modulation by phosphorylation."

No. Sentence Comment

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

PMID: 20421370 [PubMed] Tsai MF et al: "Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel."

No. Sentence Comment

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

PMID: 20657600 [PubMed] Giuliani R et al: "Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols."

No. Sentence Comment

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

PMID: 20805575 [PubMed] Bai Y et al: "Dual roles of the sixth transmembrane segment of the CFTR chloride channel in gating and permeation."

No. Sentence Comment

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

PMID: 20932301 [PubMed] Green DM et al: "Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients."

No. Sentence Comment

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

PMID: 20974851 [PubMed] Melani R et al: "Modulation of cystic fibrosis transmembrane conductance regulator (CFTR) activity and genistein binding by cytosolic pH."

No. Sentence Comment

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

PMID: 21078867 [PubMed] Kopeikin Z et al: "On the mechanism of CFTR inhibition by a thiazolidinone derivative."

No. Sentence Comment

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

PMID: 21383017 [PubMed] Pedemonte N et al: "Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations."

No. Sentence Comment

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

PMID: 21576373 [PubMed] Szollosi A et al: "Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating."

No. Sentence Comment

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

PMID: 21594775 [PubMed] Pedemonte N et al: "High-throughput screening of libraries of compounds to identify CFTR modulators."

No. Sentence Comment

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

PMID: 21658632 [PubMed] Becq F et al: "Pharmacological therapy for cystic fibrosis: from bench to bedside."

No. Sentence Comment

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

PMID: 9895335 [PubMed] Cremonesi L et al: "Validation of double gradient denaturing gradient gel electrophoresis through multigenic retrospective analysis."

No. Sentence Comment

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

PMID: 9950763 [PubMed] Clancy JP et al: "Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2B receptor-coupled pathway."

No. Sentence Comment

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

PMID: 22752155 [PubMed] Galfre E et al: "A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution."

No. Sentence Comment

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

PMID: 22927224 [PubMed] Giampieri M et al: "Asymmetric 4-Aryl-1,4-dihydropyridines Potentiate Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)."

No. Sentence Comment

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

PMID: 22889557 [PubMed] Visentin S et al: "Ligand-based design, in silico ADME-Tox filtering, synthesis and biological evaluation to discover new soluble 1,4-DHP-based CFTR activators."

No. Sentence Comment

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

PMID: 22293084 [PubMed] Yu H et al: "Ivacaftor potentiation of multiple CFTR channels with gating mutations."

No. Sentence Comment

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

PMID: 22508846 [PubMed] Jih KY et al: "Identification of a novel post-hydrolytic state in CFTR gating."

No. Sentence Comment

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

PMID: 21931512 [PubMed] Polizzi A et al: "Genotype-phenotype correlation in cystic fibrosis patients bearing [H939R;H949L] allele."

No. Sentence Comment

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

PMID: 19167254 [PubMed] Huang SY et al: "Molecular modeling of the heterodimer of human CFTR's nucleotide-binding domains using a protein-protein docking approach."

No. Sentence Comment

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

PMID: 18417076 [PubMed] Cheung JC et al: "Molecular basis for the ATPase activity of CFTR."

No. Sentence Comment

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

PMID: 16156102 [PubMed] Dunbar SA et al: "Rapid screening for 31 mutations and polymorphisms in the cystic fibrosis transmembrane conductance regulator gene by Lminex xMAP suspension array."

No. Sentence Comment

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

PMID: 15507674 [PubMed] Hadd AG et al: "Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms."

No. Sentence Comment

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

PMID: 15351355 [PubMed] Galietta LJ et al: "Identification of CFTR activators and inhibitors: chance or design?"

No. Sentence Comment

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

PMID: 15163550 [PubMed] Melin P et al: "The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein."

No. Sentence Comment

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

PMID: 9674722 [PubMed] Schwiebert EM et al: "Cystic fibrosis: a multiple exocrinopathy caused by dysfunctions in a multifunctional transport protein."

No. Sentence Comment

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

PMID: 9558482 [PubMed] Foskett JK et al: "ClC and CFTR chloride channel gating."

No. Sentence Comment

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

PMID: 10200050 [PubMed] de Meeus A et al: "Genetic findings in congenital bilateral aplasia of vas deferens patients and identification of six novel mutatations. Mutations in brief no. 138. Online."

No. Sentence Comment

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

PMID: 9511932 [PubMed] Clancy JP et al: "Purification, characterization, and expression of CFTR nucleotide-binding domains."

No. Sentence Comment

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

PMID: 9272157 [PubMed] Dork T et al: "Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens."

No. Sentence Comment

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

PMID: 8702904 [PubMed] Cotten JF et al: "Effect of cystic fibrosis-associated mutations in the fourth intracellular loop of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

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

PMID: 8662892 [PubMed] Seibert FS et al: "Disease-associated mutations in the fourth cytoplasmic loop of cystic fibrosis transmembrane conductance regulator compromise biosynthetic processing and chloride channel activity."

No. Sentence Comment

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

PMID: 8741733 [PubMed] Wilkinson DJ et al: "CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state."

No. Sentence Comment

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

PMID: 8572187 [PubMed] Howard M et al: "Epitope tagging permits cell surface detection of functional CFTR."

No. Sentence Comment

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

PMID: 7544319 [PubMed] Brancolini V et al: "Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations."

No. Sentence Comment

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

PMID: 7739684 [PubMed] Chillon M et al: "Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens."

No. Sentence Comment

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

PMID: 7534226 [PubMed] Sheppard DN et al: "Mechanism of dysfunction of two nucleotide binding domain mutations in cystic fibrosis transmembrane conductance regulator that are associated with pancreatic sufficiency."

No. Sentence Comment

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

PMID: 8825494 [PubMed] Zielenski J et al: "Cystic fibrosis: genotypic and phenotypic variations."

No. Sentence Comment

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

PMID: 7694298 [PubMed] Smit LS et al: "Functional roles of the nucleotide-binding folds in the activation of the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

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

PMID: 7686820 [PubMed] Welsh MJ et al: "Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis."

No. Sentence Comment

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

PMID: 1279852 [PubMed] Tsui LC et al: "The spectrum of cystic fibrosis mutations."

No. Sentence Comment

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

PMID: 1281034 [PubMed] Higgins CF et al: "Cystic fibrosis transmembrane conductance regulator (CFTR)."

No. Sentence Comment

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

PMID: 10762539 [PubMed] Mickle JE et al: "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."

No. Sentence Comment

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

PMID: 11448786 [PubMed] Wine JJ et al: "Cystic fibrosis: the 'bicarbonate before chloride' hypothesis."

No. Sentence Comment

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

PMID: 1375035 [PubMed] Welsh MJ et al: "Cystic fibrosis transmembrane conductance regulator: a chloride channel with novel regulation."

No. Sentence Comment

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

PMID: 14759515 [PubMed] Taddei A et al: "Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker."

No. Sentence Comment

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

PMID: 17573123 [PubMed] Amaral MD et al: "Molecular targeting of CFTR as a therapeutic approach to cystic fibrosis."

No. Sentence Comment

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

PMID: 20166764 [PubMed] Becq F et al: "Cystic fibrosis transmembrane conductance regulator modulators for personalized drug treatment of cystic fibrosis: progress to date."

No. Sentence Comment

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

PMID: 21421917 [PubMed] Balch WE et al: "Emergent properties of proteostasis in managing cystic fibrosis."

No. Sentence Comment

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

PMID: 21897819 [PubMed] Dannhoffer L et al: "Stimulation of Wild-Type, F508del- and G551D-CFTR Chloride Channels by Non-Toxic Modified pyrrolo[2,3-b]pyrazine Derivatives."

No. Sentence Comment

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

PMID: 23199563 [PubMed] Leonard A et al: "[Mucoviscidosis: CFTR mutation-specific therapy: a ray of sunshine in a cloudy sky]."

No. Sentence Comment

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

PMID: 23440202 [PubMed] Jih KY et al: "Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle."

No. Sentence Comment

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

PMID: 23620589 [PubMed] Okeyo G et al: "Converting nonhydrolyzable nucleotides to strong cystic fibrosis transmembrane conductance regulator (CFTR) agonists by gain of function (GOF) mutations."

No. Sentence Comment

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

PMID: 23757197 [PubMed] Galietta LJ et al: "Managing the underlying cause of cystic fibrosis: a future role for potentiators and correctors."

No. Sentence Comment

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

PMID: 23775370 [PubMed] Abou Tayoun AN et al: "A comprehensive assay for CFTR mutational analysis using next-generation sequencing."

No. Sentence Comment

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

PMID: 23891399 [PubMed] Van Goor F et al: "Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function."

No. Sentence Comment

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

PMID: 24030637 [PubMed] Deeks ED et al: "Ivacaftor: a review of its use in patients with cystic fibrosis."

No. Sentence Comment

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

PMID: 24420771 [PubMed] Csanady L et al: "Catalyst-like modulation of transition states for CFTR channel opening and closing: new stimulation strategy exploits nonequilibrium gating."

No. Sentence Comment

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

PMID: 24440181 [PubMed] De Boeck K et al: "The relative frequency of CFTR mutation classes in European patients with cystic fibrosis."

No. Sentence Comment

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

PMID: 24520399 [PubMed] Char JE et al: "A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor."

No. Sentence Comment

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

PMID: 24727426 [PubMed] Wang Y et al: "Understanding how cystic fibrosis mutations disrupt CFTR function: from single molecules to animal models."

No. Sentence Comment

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

PMID: 24832698 [PubMed] Balfour-Lynn IM et al: "Personalised medicine in cystic fibrosis is unaffordable."

No. Sentence Comment

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

PMID: 24932877 [PubMed] Bell SC et al: "New pharmacological approaches for cystic fibrosis: promises, progress, pitfalls."

No. Sentence Comment

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

PMID: 25033378 [PubMed] LaRusch J et al: "Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis."

No. Sentence Comment

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

PMID: 25083129 [PubMed] Pettit RS et al: "CFTR Modulators for the Treatment of Cystic Fibrosis."

No. Sentence Comment

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

PMID: 25088968 [PubMed] Chen JH et al: "A cocktail drug therapy for patients with cystic fibrosis?"

No. Sentence Comment

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

PMID: 25225552 [PubMed] Lin WY et al: "A single amino acid substitution in CFTR converts ATP to an inhibitory ligand."

No. Sentence Comment

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

PMID: 25287046 [PubMed] Mornon JP et al: "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."

No. Sentence Comment

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

PMID: 25304080 [PubMed] Dell'Edera D et al: "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."

No. Sentence Comment

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

PMID: 25675504 [PubMed] Gao X et al: "Localizing a gate in CFTR."

No. Sentence Comment

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

PMID: 25697318 [PubMed] Lay-Son R G et al: "[CFTR gene sequencing in a group of Chilean patients with cystic fibrosis]."

No. Sentence Comment

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

PMID: 25739099 [PubMed] Regan JF et al: "A rapid molecular approach for chromosomal phasing."

No. Sentence Comment

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

PMID: 25867140 [PubMed] Eckford PD et al: "Functional reconstitution and channel activity measurements of purified wildtype and mutant CFTR protein."

No. Sentence Comment

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

PMID: 25910067 [PubMed] Lucarelli M et al: "A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis."

No. Sentence Comment

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

PMID: 25940043 [PubMed] Corvol H et al: "Translating the genetics of cystic fibrosis to personalized medicine."

No. Sentence Comment

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

PMID: 26021452 [PubMed] Corvol H et al: "[Challenges of personalized medicine for cystic fibrosis]."

No. Sentence Comment

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

PMID: 26115565 [PubMed] Mall MA et al: "Targeting ion channels in cystic fibrosis."

No. Sentence Comment

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

PMID: 26526359 [PubMed] Amaral MD et al: "Hallmarks of therapeutic management of the cystic fibrosis functional landscape."

No. Sentence Comment

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