ABCMdb

ABCC7 p.His620Gln

ClinVar:	c.1859A>C , p.His620Pro ? , not provided c.1860T>G , p.His620Gln ? , not provided
CF databases:	c.1860T>G , p.His620Gln (CFTR1) D , The H620Q mutation was identified in a German patient with mild CF and who is heterozygous for this mutation and [delta]F508. c.1859A>C , p.His620Pro (CFTR1) ? , The above mutation was detected by SSCP and identified by direct DNA sequencing. H620P was found in a 15-year old male CF patient who has mild CF and who is pancreatic sufficient. His parents were unavailable for testing but one is Caucasian and the other is Asian. His mother's mutation is R1158X, which this investigators have seen in one Arabic and one Greek patient. H620P was seen only once in 100 non-[delta]F508 chromosomes screened.
Predicted by SNAP2:	A: D (85%), C: D (91%), D: D (85%), E: D (80%), F: D (95%), G: D (85%), I: D (95%), K: D (75%), L: D (91%), M: D (85%), N: D (75%), P: D (71%), Q: D (59%), R: D (85%), S: D (80%), T: D (85%), V: D (85%), W: D (95%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: N, F: D, G: D, I: D, K: D, L: D, M: D, N: N, P: D, Q: D, R: N, S: D, T: D, V: D, W: D, Y: D,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[hide] Insight in eukaryotic ABC transporter function by ... FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19. Frelet A, Klein M
Insight in eukaryotic ABC transporter function by mutation analysis.
FEBS Lett. 2006 Feb 13;580(4):1064-84. Epub 2006 Jan 19., 2006-02-13 [PMID:16442101]

Abstract [show]
With regard to structure-function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
296 G622D and R792G have reduced intrinsic chloride channel activities whereas H620Q and A800G resulted in increased intrinsic chloride transport properties [160]. Login to comment

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

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

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
50 Two sets of particularly interesting mutants are G551D and G551S and H620Q and A800G. Login to comment
54 When expressed in oocytes, the H620Q and A800G mutants increased the macroscopic Cl- current about threefold and the channel open probability by 150±180% (ref. 19). Login to comment
57 The H620Q mutant, found in one CF patient with pancreatic insuf®ciency, only stimulated HCO3 transport about 13% as effectively as did CFTR. Login to comment
66 Notably, although a few of these mutants exhibit altered letters to nature NATURE |VOL 410 |1 MARCH 2001 |www.nature.com 95 NO3 - Forskolin 5 µM NO3 - Forskolin 5 µM a G551S b G551D 10mMCl- 200 s NO3 - NO3 - Forskolin 5 µM Forskolin 5 µM f H620Q g A800G h H620Q Forskolin 5 µMCl- free Cl- freeCl- free Cl-free c G551S d G551D Forskolin 5 µM Forskolin 5 µM 0.25pHunits 250 s e A800G 0 0.25 0.50 0.75 1.00 H620Q A800G G551D G551S j WT Forskolin 5 µM 0 0.25 0.50 0.75 1.00 H620Q A800G G551D G551S WT i [Cl- ]change(mMs-1 )HCO3 -transport (∆pH+ min-1 ) Figure 2 cAMP-stimulated Cl- and HCO3 transport by CFTR mutants associated with a severe or a mild form of CF. Login to comment
186 letters to nature 96 NATURE |VOL 410 |1 MARCH 2001 |www.nature.com HCO3 -/Cl- transportratio 0 0.25 0.50 0.75 1.00 WT I148T G178R R297Q G551D H620Q G970R A1067T G1244E S1255P G1349D E193K G551S A800G H949Y R1070Q Pancreatic insufficient Pancreatic sufficientD648V N CI148T G178R E193K R297Q R117H A1067T R1070Q G1244E S1255P G1349D NBD2 RD H949Y G970R CL4CL3CL2CL1 NBD1 G551D G551S H620Q D648V A800G Figure 3 The HCO3:Cl-transport ratio of CFTR mutants associated with CF. Login to comment

[hide] The phenotypic consequences of CFTR mutations. Ann Hum Genet. 2003 Sep;67(Pt 5):471-85. Rowntree RK, Harris A
The phenotypic consequences of CFTR mutations.
Ann Hum Genet. 2003 Sep;67(Pt 5):471-85., [PMID:12940920]

Abstract [show]
Cystic fibrosis is a common autosomal recessive disorder that primarily affects the epithelial cells in the intestine, respiratory system, pancreas, gall bladder and sweat glands. Over one thousand mutations have currently been identified in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that are associated with CF disease. There have been many studies on the correlation of the CFTR genotype and CF disease phenotype; however, this relationship is still not well understood. A connection between CFTR genotype and disease manifested in the pancreas has been well described, but pulmonary disease appears to be highly variable even between individuals with the same genotype. This review describes the current classification of CFTR mutation classes and resulting CF disease phenotypes. Complex disease alleles and modifier genes are discussed along with alternative disorders, such as disseminated bronchiectasis and pancreatitis, which are also thought to result from CFTR mutations.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
78 Three mutant CFTR proteins, G622D, R792G and E822K, that were transiently expressed in COS cells showed lower chloride channel activities when compared to wild-type CFTR, whereas mutants H620Q and A800G showed increased activities. Login to comment

[hide] Nucleotide binding domains of human CFTR: a struct... Cell Mol Life Sci. 2005 Sep;62(18):2112-23. Eudes R, Lehn P, Ferec C, Mornon JP, Callebaut I
Nucleotide binding domains of human CFTR: a structural classification of critical residues and disease-causing mutations.
Cell Mol Life Sci. 2005 Sep;62(18):2112-23., [PMID:16132229]

Abstract [show]
Defective function of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes CF, the most frequent lethal inherited disease among the Caucasian population. The structure of this chloride ion channel includes two nucleotide-binding domains (NBDs), whose ATPase activity controls channel gating. Recently, the experimental structures of mouse and human CFTR NBD1 and our model of the human CFTR NBD1/NBD2 heterodimer have provided new insights into specific structural features of the CFTR NBD dimer. In the present work, we provide a structural classification of CF-causing mutations which may complement the existing functional classification. Our analysis also identified amino acid residues which may play a critical role in interdomain interaction and are located at the NBD1-NBD2 interface or on the surface of the dimer. In particular, a cluster of aromatic amino acids, which includes F508 and straddles the two NBDs, might be directly involved in the interaction of the NBD1/NBD2 heterodimer with the channel-forming membrane-spanning domains.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
247 (B) Ribbon representation of the NBD aa1 - aa2 segment of the three experimental MalK structures and of mCFTR NBD1, with the CFTR F508 and MalK F98 being shown in atomic details involves the exposed F508 residue, only a few missense mutations affecting these exposed aromatic residues have been reported: H484R, Y515H, H620P, H620Q and Y1307C (http://www.genet.sickkids.on.ca/cftr). Login to comment

[hide] Too much salt, too little soda: cystic fibrosis. Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415. Quinton PM
Too much salt, too little soda: cystic fibrosis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415., 2007-08-25 [PMID:17700961]

Abstract [show]
Cystic fibrosis (CF) of the pancreas is the most widely accepted name of the most common fatal inherited single gene defect disease among Caucasians. Its incidence among other races is thought to be significantly less, but mutations in the gene have been reported in most, if not all, major populations. This review is intended to give general concepts of the molecular as well as physiological basis of the pathology that develops in the disease. First, an overview of the organ pathology and genetics is presented, followed by the molecular structure of the gene product (cystic fibrosis transmembrane conductance regulator, CFTR), its properties, functions, and controls as currently understood. Second, since mutations appear to be expressed primarily as a defect in electrolyte transport, effects and mechanisms of pathology are presented for two characteristically affected organs where the etiology is best described: the sweat gland, which excretes far too much NaCl ("salt") and the pancreas, which excretes far too little HCO3(- )("soda"). Unfortunately, morbidity and mortality in CF develop principally from refractory airway infections, the basis of which remains controversial. Consequently, we conclude by considering possible mechanisms by which defects in anion transport might predispose the CF lung to chronic infections.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
177 The activity was CFTR mutation-specific and, for example, mutations such as R117H (the pancreas is spared in compound CF heterozygotes such as R117H/ΔF508)appeared to retain the ability to support exchange even though Cl-conductance was depressed while mutations such as H620Q did not support exchange, but retained a disputed[240] Cl-conductance. Login to comment

[hide] C terminus of nucleotide binding domain 1 contains... J Biol Chem. 2010 Jul 16;285(29):22132-40. Epub 2010 Apr 30. Billet A, Melin P, Jollivet M, Mornon JP, Callebaut I, Becq F
C terminus of nucleotide binding domain 1 contains critical features for cystic fibrosis transmembrane conductance regulator trafficking and activation.
J Biol Chem. 2010 Jul 16;285(29):22132-40. Epub 2010 Apr 30., 2010-07-16 [PMID:20435887]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest beta strands (beta(c)5 and beta(c)6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the beta(c)5-beta(c)6 hairpin, within the beta(c)5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the beta(c)6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
2 We mutated CFTR amino acids located in the betac5-betac6 hairpin, within the betac5 strand (H620Q), within the beta-turn linking the two beta strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the betac6 strand. Login to comment
3 Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. Login to comment
5 In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Login to comment
33 We have mutated these two glycine residues in aspartic acid (G622D) and arginine (G628R) and considered other mutants in their neighborhood (H620Q, E621G, S623A, and S624A). Login to comment
87 RESULTS Expression of the NBD1 C-terminal CFTR Mutants-We have introduced EGFP-tagged CFTR proteins into HEK293 cells, wt CFTR and six CFTR mutants, i.e. H620Q, E621G, G622D, S623A, S624A, and G628R. Login to comment
90 For the expression of CFTR mutants H620Q, E621G, S623A, and S624A, the profiles of core-glycosylated and mature-glycosylated forms were similar to that of the wt protein. Login to comment
109 Second, with the two mutations introduced on both sides of the beta turn, H620Q and S623A, we recorded an increased (p Ͻ 0.001) Cl- current (supplemental Table 2) compared with wt CFTR. Login to comment
122 Concerning the activation kinetics, only the two CFTR mutants with an increased Cl-transport activity (H620Q and S623A) also present a faster time course of activation (Fig. 5B). Login to comment
124 T50 of the two mutated channels (T50 ϭ 124.45 Ϯ 8.8 s for H620Q, n ϭ 8 and T50 ϭ 114.63 Ϯ 8.8 s for S623A, n ϭ 8) were significantly different compared with wt (T50 ϭ 164.85 Ϯ 10.2 s, n ϭ 8). Login to comment
125 Our results indicated that H620Q and S623A channels could be activated more rapidly than the other CFTR mutant channels studied. Login to comment
130 Interestingly, although the level of current for H620Q and S623A was greater than the response of wt channels (Fig. 3), both mutated CFTRs were activated by MPB-91 to a level similar to that of wt CFTR (Fig. 6) (at 40 mV, current densities were: H620Q, 27.86 Ϯ 4.2 pA/pF, n ϭ 5; S623A, 30.83 Ϯ 3.4 pA/pF, n ϭ 6). Login to comment
163 In particular, the cAMP-activated Cl- current densities elicited by H620Q and S623A channels were increased compared with wt. Login to comment
165 For H620Q mutant, this increase could be explained by the reported increased of open probability (15). Login to comment
169 The H620Q mutation might thus disturb the contacts existing between the beta hairpin and the P-loop of NBD1 and thus might result in a perturbation of the channel gating through an effect on the ATP binding and/or hydrolysis in the noncanonical site. Login to comment
180 In addition, MPB-91 was also able to activate H620Q, E621G, S623A, and S624A mutants. Login to comment
182 Contrary to the activation by Fsk, no difference of kinetic parameters was detected with the two mutants H620Q and S623A. Login to comment

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

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

Comments [show]

None has been submitted yet.

Sentences [show]
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

[hide] Characterization of 19 disease-associated missense... Hum Mol Genet. 1998 Oct;7(11):1761-9. Vankeerberghen A, Wei L, Jaspers M, Cassiman JJ, Nilius B, Cuppens H
Characterization of 19 disease-associated missense mutations in the regulatory domain of the cystic fibrosis transmembrane conductance regulator.
Hum Mol Genet. 1998 Oct;7(11):1761-9., [PMID:9736778]

Abstract [show]
In order to gain a better insight into the structure and function of the regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, 19 RD missense mutations that had been identified in patients were functionally characterized. Nine of these (I601F, L610S, A613T, D614G, I618T, L619S, H620P, G628R and L633P) resulted in aberrant processing. No or a very small number of functional CFTR proteins will therefore appear at the cell membrane in cells expressing these mutants. These mutations were clustered in the N-terminal part of the RD, suggesting that this subdomain has a folding pattern that is very sensitive to amino acid changes. Mutations that caused no aberrant processing were further characterized at the electrophysiological level. First, they were studied at the whole cell level in Xenopus laevis oocytes. Mutants that induced a whole cell current that was significantly different from wild-type CFTR were subsequently analysed at the single channel level in COS1 cells transiently expressing the different mutant and wild-type proteins. Three mutant chloride channels, G622D, R792G and E822K CFTR, were characterized by significantly lower intrinsic chloride channel activities compared with wild-type CFTR. Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Finally, T665S and E826K CFTR had single channel properties not significantly different from wild-type CFTR.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
8 Two mutations, H620Q and A800G, resulted in increased intrinsic chloride transport activities. Login to comment
68 Primers used for mutagenesis Primer Sequence I601F (a1933t) 5'-CTA ACA AAA CTA GGT TTT TGG TCA CTT C-3' L610S (t1961c) 5'-CTA AAA TGG AAC ATT CAA AGA AAG CTG-3' A613T (g1969a) 5'-CAT TTA AAG AAA ACT GAC AAA ATA TTA-3' D614G (a1973g) 5'-CAT TTA AAG AAA GCT GGC AAA ATA TTA A-3' I618T (t1985c) 5'-GAC AAA ATA TTA ACT TTG CAT GAA GG-3' L619S (t1988c) 5'-GAC AAA ATA TTA ATT TCG CAT GAA GGT-3' H620P (a1991c) 5'-CAA AAT ATT AAT TTT GCC TGA AGG TAG C-3' H620Q (t1992g) 5'-AAT ATT AAT TTT GCA GGA AGG TAG CAG-3' G622D (g1997a) 5'-TTG CAT GAA GAT AGC AGC TAT TTT TAT G-3' G628R (g2014c) 5'-GCA GCT ATT TTT ATC GGA CAT TTT C-3' L633P (t2030c) 5'-CAT TTT CAG AAC CCC AAA ATC TAC AGC-3' D648V (a2075t) 5'-CTC ATG GGA TGT GTT TCT TTC GAC C-3' T665S (a2125t) 5'-CAA TCC TAA CTG AGT CCT TAC ACC G-3' F693L (t2209c) 5'-CAG ACT GGA GAG CTT GGG GAA AAA AG-3' R766M (g2429t) 5'-GCA CGA AGG ATG CAG TCT GTC CTG-3' R792G (c2506g) 5'-CAG CAT CCA CAG GAA AAG TGT CAC TG-3' A800G (c2531g) 5'-CTG GCC CCT CAG GGA AAC TTG ACT G-3' I807M (a2553g) 5'-CTG AAC TGG ATA TGT ATT CAA GAA GG-3' E822K (g2596a) 5'-GGC TTG GAA ATA AGT AAA GAA ATT AAC G-3' E826K (g2608a) 5'-GAA GAA ATT AAC AAA GAA GAC TTA AAG-3' Selection primer BstBI 5'-CTC TGG GGT CCG GAA TGA CCG AC-3' Two primers were used for each mutagenesis reaction. Login to comment
77 Mutations detected in patients (I601F, L610S, A613T, D614G, I618T, L619S, H620P, H620Q, D622G, G628R, L633P, T665S, F693L, K698R, V754M, R766M, R792G, A800G, I807M, E822K and E826K) are indicated in bold and underlined, the PKA phosphorylation sites by an arrow and the two acidic domains are boxed. Login to comment
84 Interestingly, two mutations (H620Q and A800G) gave rise to chloride channels with significantly higher chloride transport activities. Login to comment
87 Maturation pattern of RD mutations and their associated phenotype found in patients with the indicated genotype (when the mutation is associated with CF, only the pancreas status is given) Mutation A-form B-form C-form Clinical data Genotype Phenotype Reference I601F + + - I601F/G542X PS M. Schwarz, personal communication L610S + + - Unknown Unknown A613T + + - Unknown Unknown D614G + + - D614G/unknown PI 14 I618T + + - I618T/dF508 PS G.R. Cutting, personal communication L619S + + - L619S/unknown PI B. Tümmler, personal communication H620P + + - H620P/R1158X PS M. Schwarz, personal communication H620Q + + + H620Q/dF508 PI T. Dörk, personal communication G622D + + + G622D/unknown Oligospermia J. Zielenski, personal communication G628R + + - Unknown Unknown L633P + + - L633P/3659delC M. Schwarz, personal communication D648V + + + D648V/3849+10kb C/T PI C. Ferec, personal communication T665S + + + Unknown Unknown F693L + + + F693L/W1282X Healthy C. Ferec; CF Genetic Analysis Consortium R766M + + + R766M/R792G CBAVD D. Glavac, personal communication R792G + + + R766M/R792G CBAVD D. Glavac, personal communication A800G + + + A800G/unknown CBAVD 34 I807M + + + I807M/unknown CBAVD Our observation E822K + + + E822K/unknown PI 35 E826K + + + E826K/unknown Thoracic sarcoidosis C. Bombieri, personal communication +, the protein matures up to that form; -, the protein does not reach the respective maturation step. Login to comment
97 G622D, R792G and E822K gave rise to a CFTR chloride channel with a significantly lower Po than wild-type CFTR; H620Q and A800G CFTR resulted in channels with significantly higher Po. Login to comment
114 When His620 was changed to Gln, noeffectonmaturationwasobserved.WhenHis620waschanged to Pro, an amino acid that dramatically affects the secondary structure of the protein and therefore possibly its correct folding, a non-maturing protein was obtained. Login to comment
123 Mutations that did not affect maturation (H620Q, G622D, D648V, T665S, F693L, R766M, R792G, A800G, I807M, E822K and E826K) were subsequently analysedat theelectrophysiologi- cal level. Login to comment
127 Strikingly, two mutations (H620Q and A800G) showed a significantly increased Po, when compared with wild-type CFTR. Login to comment
130 The 'hyperactive` disease-causing mutations described here, H620Q and A800G, do mature. Login to comment
136 Activation of single channels by expressing wild-type and mutant CFTR (H620Q). Login to comment
137 (A) Single channel current tracings of wild-type and H620Q CFTR before and after addition of the activation cocktail (1 µM IBMX and 0.1 µM forskolin) to the external site of the membrane patch. Login to comment

[hide] Conformational changes relevant to channel activit... J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21. Hudson RP, Chong PA, Protasevich II, Vernon R, Noy E, Bihler H, An JL, Kalid O, Sela-Culang I, Mense M, Senderowitz H, Brouillette CG, Forman-Kay JD
Conformational changes relevant to channel activity and folding within the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2012 Aug 17;287(34):28480-94. doi: 10.1074/jbc.M112.371138. Epub 2012 Jun 21., [PMID:22722932]

Abstract [show]
Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between beta-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with beta-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
1 Results: H620Q mutation associated with increased channel Po, and the corrector/potentiator CFFT-001 both lead to similar conformational shifts in NBD1. Login to comment
5 NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between beta-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. Login to comment
7 Decreases in Tm from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. Login to comment
50 To probe the conformational effects of mutations and binding of small molecule modulators, we have applied a range of biophysical approaches to study H620Q and helix H9 variants of NBD1 and the interaction of the most soluble compound of a series of dual corrector/potentiator compounds provided by the Cystic Fibrosis Foundation, N-cyclohexyl-4-(6-methyl-3-pyridinyl) pyrimidine-2-amine, referred to here as CFFT-001. Login to comment
51 NMR backbone resonance assignments (82%) carried out on a human F508del NBD1 lacking the RI and ending at residue 646 (F508del NBD1 ⌬RI⌬RE) enable us to define conformational changes due to the H620Q mutation. Login to comment
53 Addition of CFFT-001 to F508del NBD1 ⌬RI⌬RE results in NBD1 conformational changes overlapping with those observed for the H620Q mutant protein that has higher open channel probability. Login to comment
54 Differential scanning calorimetry (DSC) data show a reduction in NBD1 thermal melting temperature for H620Q or in the presence of CFFT-001, demonstrating direct binding of the compound to a less thermostable conformation. Login to comment
57 EXPERIMENTAL PROCEDURES Protein Expression and Purification (NMR and DSC Studies)- Human NBD1 (387-646, ⌬405-436) constructs with or without Phe-508 and containing the H620Q mutation or deletion of helix H9(636-646) were expressed as His6-SUMO fusions at 16 °C in BL21(DE3) Codon Plus cells grown in minimal media with [15 N]NH4Cl and/or [13 C]glucose, for NMR studies, or LB for DSC studies, and purified as described previously (31, 32). Login to comment
97 Repeated measurements for the H620Q F508del sample gave identical Tm values to the first decimal place; the general reproducibility of NBD1 Tm values is within Ϯ0.3 °C. Login to comment
116 H620Q Variant Reduces Helicity of H8 and H9 at the C Terminus of F508del NBD1 ⌬RI⌬RE-To address possible conformational changes within NBD1 that may be relevant to channel activity and/or misfolding, we focused on the C-terminal beta-strands (S9 and S10) which have been shown to affect processing, activity and pharmacology of CFTR (56). Login to comment
117 In particular, an H620Q variant (in S9) originally identified in CF patients has an increased Po in single channels (56-58). Login to comment
118 Fig. 2, A and B, presents an overlay of NMR spectra for F508del NBD1 ⌬RI⌬RE and its H620Q variant (F508del H620Q NBD1 ⌬RI⌬RE). Login to comment
119 Arrows indicate a subset of peaks that shift upon H620Q mutation, including Glu-621, Gly-622, Gln-634, Leu-636, Ser-641, Leu-644, and Met-645. Login to comment
134 Chemical shifts in the absence of the mutation demonstrate that these helices are in equilibrium with a coil conformation (supplemental Fig. S4C) (59) and the chemical shift perturbations for the residues of the H8/H9 helices upon replacing His-620 with a Gln reflect a change in the equilibrium further toward the coil conformation. Login to comment
142 H620Q variant reduces helicity of H8 and H9 at the C terminus of F508del NBD1 ⌬RI⌬RE. Login to comment
143 A, overlay of 15 N-1 H correlation spectra at 500 MHz for F508del NBD1 ⌬RI⌬RE (black; background) and the H620Q variant, F508del H620Q NBD1 ⌬RI⌬RE (red; foreground). Login to comment
145 Asterisks show a separate subset of peaks that shift in the H620Q variant but do not shift upon addition of CFFT-001. Login to comment
147 C, same overlay as in B, with a third layer showing the addition of compound to the H620Q variant (green; foreground). Login to comment
149 E, ribbon diagram of WT NBD1 ⌬RI⌬RE (2PZE) with unassigned residues in cyan, and assigned residues that do not shift upon H620Q mutation are shown in light gray. Login to comment
161 Comparison of this set of peaks with a subset of those resulting from the H620Q mutation (compare Fig. 2 with Fig. 4) shows significant similarities, including both the identity of perturbed resonances and the direction of the chemical shift changes. Login to comment
162 These similarities are consistent with a common effect of the H620Q substitution and CFFT-001 on H8 and H9, although additional peak shifts reflecting other conformational changes in the mutant are present as well (Fig. 2A). Login to comment
165 Similar to the H620Q variant, residues on both surfaces of these helices are affected by CFFT-001, as opposed to one surface of the helix as one might expect for a direct drug interaction with the helix. Login to comment
197 Interestingly, titration of the compound into the F508del H620Q NBD1 ⌬RI⌬RE leads to resonances of H9 shifting more toward coil (Fig. 2C, arrows). Login to comment
198 Thus, the compound further pushes the conformational equilibrium shift already present in the H620Q variant, in a qualitatively additive fashion, and the mutation does not inhibit compound binding. Login to comment
227 In all of these binding modes, interaction of CFFT-001 with the surface of strands S3, S9, and S10 is not expected to be significantly perturbed by mutation of H620Q, which is adjacent to but not directly at the proposed binding surface (Fig. 6 and supplemental Fig. S6). Login to comment
230 The data in Fig. 7A illustrate that both the H620Q variant and the deletion of H9 reduce the thermal stability of NBD1. Login to comment
231 The midpoint of thermal denaturation, Tm, is reduced by ϳ1-2 degrees in each, comparing F508del NBD1 ⌬RI⌬RE (Tm in 2 mM ATP ϭ 49.4 °C and 5 mM ATP ϭ 51.0 °C) to either F508del H620Q NBD1 ⌬RI⌬RE (bottom curve, Tm in 2 mM ATP ϭ 47.1 °C) or F508del NBD1 ⌬RI⌬H9 (middle curve, Tm in 5 mM ATP ϭ 50.4 °C). Login to comment
244 Overall, the DSC data are in agreement with the effects of deletion of H9, an H620Q variant, and addition of CFFT-001 on NBD1 inferred from NMR data for these constructs. Login to comment
250 A, DSC traces for WT and F508del NBD1 ⌬RI⌬RE (upper curves) in the absence (solid lines) and presence (dashed lines) of CFFT-001; buffer includes 2 mM ATP. DSC traces for F508del NBD1 ⌬RI⌬H9 (middle curves) in the absence (solid line) and presence (dashed line) of CFFT-001; buffer includes 5 mM ATP. DSC traces for F508del H620Q NBD1 ⌬RI⌬RE (lower curve); buffer includes 2 mM ATP. Login to comment
264 NMR has enabled us to probe these effects at a residue-specific level and demonstrate that the H620Q substitution associated with higher channel open probability and a dual corrector/potentiator compound give rise to similar conformational changes within NBD1. Login to comment
266 The significance of the DSC results for the H620Q and H9 deletion can be better appreciated in the context of our current understanding of the NBD1 thermal unfolding pathway derived from a comprehensive analysis of previous DSC data on WT and F508del NBD1 (32, 64). Login to comment
268 The H620Q and ⌬H9 mutations also may reduce the thermodynamic stability of NBD1, or accelerate the rate of aggregation, or both. Login to comment
283 The H620Q variant, originally identified in CF patients demonstrating pancreatic insufficiency, has previously been shown to increase the Po of single CFTR channels, indicating an effect on the gating properties of CFTR (56-58). Login to comment
284 Both the H620Q variant and the CFFT-001 compound cause a shift of helices H8 and H9 from a preexisting helix-coil conformational equilibrium toward the coil state. These results suggest that perturbations affecting this conformational equilibrium coincide with conditions that promote channel opening and/or impede channel closing. Login to comment
285 Because the H620Q variant displays additional peak shifts relative to those observed for compound binding, there are certainly other consequences of the mutation. Login to comment
287 Although H620Q does not affect maturation/processing, an H620P mutation does, highlighting the importance of this position and the nature of the residue in it. Login to comment
294 Thus, we hypothesize that conformational changes, as elicited by H620Q or CFFT-001, result in a shift in an underlying conformational equilibrium of regulatory interactions that are normally involved in gating. Login to comment
296 This should promote NBD dimerization and lead to an enhanced open probability, increased channel activity for H620Q mutant channels and the potentiating effect of CFFT-001. Login to comment
297 It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Login to comment
305 Addition of compound or the H620Q mutation shifts this equilibrium by reducing H9 helicity and contacts with the NBD1, subsequently leading to release of the RE/R region from the dimerization interface, relieving the inhibition and facilitating NBD1/NBD2 heterodimerization and channel opening. Login to comment
316 The large number of substitutions in NBD1 that can suppress the F508del mutation supports such a general allosteric view of NBD1 with the structural changes we observe in the H620Q variant and upon CFFT-001 binding being one part of the NBD1 conformational equilibria. Login to comment
320 Because the H620Q mutation and CFFT-001 are both associated with an increase in the open probability of CFTR channels and because H8/H9 lead directly into the RE/R region within the context of full-length CFTR, we have hypothesized that this conformational shift at H8/H9 releases the R region from the NBD1/ NBD2 dimerization interface, allowing heterodimers to form and thereby enhancing channel open probability and possibly processing. Login to comment
48 To probe the conformational effects of mutations and binding of small molecule modulators, we have applied a range of biophysical approaches to study H620Q and helix H9 variants of NBD1 and the interaction of the most soluble compound of a series of dual corrector/potentiator compounds provided by the Cystic Fibrosis Foundation, N-cyclohexyl-4-(6-methyl-3-pyridinyl) pyrimidine-2-amine, referred to here as CFFT-001. Login to comment
49 NMR backbone resonance assignments (82%) carried out on a human F508del NBD1 lacking the RI and ending at residue 646 (F508del NBD1 èc;RIèc;RE) enable us to define conformational changes due to the H620Q mutation. Login to comment
52 Differential scanning calorimetry (DSC) data show a reduction in NBD1 thermal melting temperature for H620Q or in the presence of CFFT-001, demonstrating direct binding of the compound to a less thermostable conformation. Login to comment
55 EXPERIMENTAL PROCEDURES Protein Expression and Purification (NMR and DSC Studies)- Human NBD1 (387-646, èc;405-436) constructs with or without Phe-508 and containing the H620Q mutation or deletion of helix H9(636-646) were expressed as His6-SUMO fusions at 16 &#b0;C in BL21(DE3) Codon Plus cells grown in minimal media with [15 N]NH4Cl and/or [13 C]glucose, for NMR studies, or LB for DSC studies, and purified as described previously (31, 32). Login to comment
141 H620Q variant reduces helicity of H8 and H9 at the C terminus of F508del NBD1 èc;RIèc;RE. Login to comment
144 Asterisks show a separate subset of peaks that shift in the H620Q variant but do not shift upon addition of CFFT-001. Login to comment
146 C, same overlay as in B, with a third layer showing the addition of compound to the H620Q variant (green; foreground). Login to comment
148 E, ribbon diagram of WT NBD1 èc;RIèc;RE (2PZE) with unassigned residues in cyan, and assigned residues that do not shift upon H620Q mutation are shown in light gray. Login to comment
160 Comparison of this set of peaks with a subset of those resulting from the H620Q mutation (compare Fig. 2 with Fig. 4) shows significant similarities, including both the identity of perturbed resonances and the direction of the chemical shift changes. Login to comment
164 Similar to the H620Q variant, residues on both surfaces of these helices are affected by CFFT-001, as opposed to one surface of the helix as one might expect for a direct drug interaction with the helix. Login to comment
196 Interestingly, titration of the compound into the F508del H620Q NBD1 èc;RIèc;RE leads to resonances of H9 shifting more toward coil (Fig. 2C, arrows). Login to comment
225 In all of these binding modes, interaction of CFFT-001 with the surface of strands S3, S9, and S10 is not expected to be significantly perturbed by mutation of H620Q, which is adjacent to but not directly at the proposed binding surface (Fig. 6 and supplemental Fig. S6). Login to comment
228 The data in Fig. 7A illustrate that both the H620Q variant and the deletion of H9 reduce the thermal stability of NBD1. Login to comment
229 The midpoint of thermal denaturation, Tm, is reduced by b03;1-2 degrees in each, comparing F508del NBD1 èc;RIèc;RE (Tm in 2 mM ATP afd; 49.4 &#b0;C and 5 mM ATP afd; 51.0 &#b0;C) to either F508del H620Q NBD1 èc;RIèc;RE (bottom curve, Tm in 2 mM ATP afd; 47.1 &#b0;C) or F508del NBD1 èc;RIèc;H9 (middle curve, Tm in 5 mM ATP afd; 50.4 &#b0;C). Login to comment
242 Overall, the DSC data are in agreement with the effects of deletion of H9, an H620Q variant, and addition of CFFT-001 on NBD1 inferred from NMR data for these constructs. Login to comment
248 A, DSC traces for WT and F508del NBD1 èc;RIèc;RE (upper curves) in the absence (solid lines) and presence (dashed lines) of CFFT-001; buffer includes 2 mM ATP. DSC traces for F508del NBD1 èc;RIèc;H9 (middle curves) in the absence (solid line) and presence (dashed line) of CFFT-001; buffer includes 5 mM ATP. DSC traces for F508del H620Q NBD1 èc;RIèc;RE (lower curve); buffer includes 2 mM ATP. Login to comment
262 NMR has enabled us to probe these effects at a residue-specific level and demonstrate that the H620Q substitution associated with higher channel open probability and a dual corrector/potentiator compound give rise to similar conformational changes within NBD1. Login to comment
281 The H620Q variant, originally identified in CF patients demonstrating pancreatic insufficiency, has previously been shown to increase the Po of single CFTR channels, indicating an effect on the gating properties of CFTR (56-58). Login to comment
282 Both the H620Q variant and the CFFT-001 compound cause a shift of helices H8 and H9 from a preexisting helix-coil conformational equilibrium toward the coil state. These results suggest that perturbations affecting this conformational equilibrium coincide with conditions that promote channel opening and/or impede channel closing. Login to comment
292 Thus, we hypothesize that conformational changes, as elicited by H620Q or CFFT-001, result in a shift in an underlying conformational equilibrium of regulatory interactions that are normally involved in gating. Login to comment
295 It is possible that H620Q and CFFT-001 act to force an "unnatural" gating; however, the likelihood of this is low, considering that the portion of NBD1 affected appears to be a regulatory hot spot based on our sequence analysis and the observed effects of mutations here (56). Login to comment
303 Addition of compound or the H620Q mutation shifts this equilibrium by reducing H9 helicity and contacts with the NBD1, subsequently leading to release of the RE/R region from the dimerization interface, relieving the inhibition and facilitating NBD1/NBD2 heterodimerization and channel opening. Login to comment
314 The large number of substitutions in NBD1 that can suppress the F508del mutation supports such a general allosteric view of NBD1 with the structural changes we observe in the H620Q variant and upon CFFT-001 binding being one part of the NBD1 conformational equilibria. Login to comment
318 Because the H620Q mutation and CFFT-001 are both associated with an increase in the open probability of CFTR channels and because H8/H9 lead directly into the RE/R region within the context of full-length CFTR, we have hypothesized that this conformational shift at H8/H9 releases the R region from the NBD1/ NBD2 dimerization interface, allowing heterodimers to form and thereby enhancing channel open probability and possibly processing. Login to comment

[hide] Mechanism of direct bicarbonate transport by the C... J Cyst Fibros. 2009 Mar;8(2):115-21. Epub 2008 Nov 18. Tang L, Fatehi M, Linsdell P
Mechanism of direct bicarbonate transport by the CFTR anion channel.
J Cyst Fibros. 2009 Mar;8(2):115-21. Epub 2008 Nov 18., [PMID:19019741]

Abstract [show]
BACKGROUND: CFTR contributes to HCO(3)(-) transport in epithelial cells both directly (by HCO(3)(-) permeation through the channel) and indirectly (by regulating Cl(-)/HCO(3)(-) exchange proteins). While loss of HCO(3)(-) transport is highly relevant to cystic fibrosis, the relative importance of direct and indirect HCO(3)(-) transport it is currently unknown. METHODS: Patch clamp recordings from membrane patches excised from cells heterologously expressing wild type and mutant forms of human CFTR were used to isolate directly CFTR-mediated HCO(3)(-) transport and characterize its functional properties. RESULTS: The permeability of HCO(3)(-) was approximately 25% that of Cl(-) and was invariable under all ionic conditions studied. CFTR-mediated HCO(3)(-) currents were inhibited by open channel blockers DNDS, glibenclamide and suramin, and these inhibitions were affected by mutations within the channel pore. Cystic fibrosis mutations previously associated with disrupted cellular HCO(3)(-) transport did not affect direct HCO(3)(-) permeability. CONCLUSIONS: Cl(-) and HCO(3)(-) share a common transport pathway in CFTR, and selectivity between Cl(-) and HCO(3)(-) is independent of ionic conditions. The mechanism of transport is therefore effectively identical for both ions. We suggest that mutations in CFTR that cause cystic fibrosis by selectively disrupting HCO(3)(-) transport do not impair direct CFTR-mediated HCO(3)(-) transport, but may predominantly alter CFTR regulation of other HCO(3)(-) transport pathways.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
101 Fig. 5 shows macroscopic currents carried by three of these mutants, two associated with loss of cellular HCO3 - transport and PI disease (G178R, H620Q) and one with retained HCO3 - transport and PS disease (G551 S) [10]. Login to comment
103 It can be seen that each of these mutants is capable of mediating HCO3 - efflux; in fact, reversal potential measurements indicate PHCO3/PCl values of 0.289±0.038 (n=3) for G178R, 0.264±0.035 (n=3) for G551S, and 0.237±0.033 (n=3) for H620Q, none of which were significantly different from the value of 0.250±0.037 (n=4) estimated from wild type under the same conditions (see Fig. 1B). Login to comment
138 Bicarbonate permeability of CF mutant forms of CFTR Example leak-subtracted macroscopic I-V relationships for G178R, G551S, and H620Q-CFTR under the same ionic conditions used in Fig. 1B. Login to comment
146 Our direct measurements of CFTR HCO3 - currents also showed no change in HCO3 - permeability in three CF-associated CFTR mutants (G178R, G551S, H620Q; Fig. 5) that were previously associated with different effects on cellular HCO3 - transport relative to Cl- transport [10]. Login to comment
147 This result suggests that loss of direct HCO3 - permeation through the channel is not responsible for the selective loss of HCO3 - transport relative to Cl- transport previously associated with the severe, PI mutations G178R and H620Q. Login to comment

[hide] Phosphorylation site independent single R-domain m... FEBS Lett. 1998 Nov 13;439(1-2):121-6. Wei L, Vankeerberghen A, Cuppens H, Droogmans G, Cassiman JJ, Nilius B
Phosphorylation site independent single R-domain mutations affect CFTR channel activity.
FEBS Lett. 1998 Nov 13;439(1-2):121-6., [PMID:9849891]

Abstract [show]
We investigated CFTR channel activity of mature R-domain mutants showing single alterations at sites other than the predicted phosphorylation sites. All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. The anion permeability sequence for all three mutants was the same as that of wild type CFTR. Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Single channel conductances were unchanged in all mutants. Our results suggest that additional sites in the R-domain other than phosphorylation sites influence gating of CFTR channels.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
1 All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). Login to comment
2 The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. Login to comment
4 Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Login to comment
25 Three di¡erent mutations, t1992g (= H620Q), g2596a (= E822K) and g2608a (= E826K), were introduced using the Transformer Site-Directed Mutagenesis kit (Clontech). Login to comment
76 COS cells were transfected with wt CFTR, E822K CFTR (top), H620Q CFTR (bottom) and E826K CFTR (bottom) and selected for 2 weeks with G418. Login to comment
88 In this study, the maturation pattern of three mutant R-domain proteins (H620Q-CFTR, E822K-CFTR and E826K-CFTR) has been characterized. Login to comment
92 Whole cells currents of mutants in Xenopus oocytes Whole cell membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild type or mutant (H620Q, E822K, E826K) constructs. Login to comment
101 Oocytes expressing the mutant R-domain protein (H620Q), in which a predominantly positively charged histidine was substituted by a less charged glutamine (at pH 7.2), showed a much larger conductance activated by application of phos-cock. Login to comment
104 The conductance activated by phos-cock in H620Q expressed cells was 27.84 þ 5.7 WS (n = 6). Login to comment
134 We found that the open probability of the E822K and E826K mutants was signi'cantly lower than that of wild type CFTR, whereas that of the H620Q mutant was strongly enhanced compared to wild type (not shown). Login to comment
136 The average number of activated channels is 2.6 þ 0.306 (n = 10) for wild-type CFTR, 3.51 þ 0.428 (n = 6) for H620Q, 1.0 þ 0.000 (n = 4) for E822K and 1.66 þ 0.211 (n = 6) for E826K. Login to comment
145 A comparison of the three R-domain mutants leads to a remarkable conclusion: both mutations, E822K and E826K, in which negatively charged glutamic acids were replaced by positively charged lysine had a signi'cantly reduced CFTR channel activity, whereas the H620Q mutation, in which positively charged histidine was replaced by the more neutral amino acid glutamine, had a much higher channel activity. Login to comment
74 COS cells were transfected with wt CFTR, E822K CFTR (top), H620Q CFTR (bottom) and E826K CFTR (bottom) and selected for 2 weeks with G418. Login to comment
86 In this study, the maturation pattern of three mutant R-domain proteins (H620Q-CFTR, E822K-CFTR and E826K-CFTR) has been characterized. Login to comment
90 Whole cells currents of mutants in Xenopus oocytes Whole cell membrane currents were recorded from Xenopus oocytes injected with RNA transcribed from either wild type or mutant (H620Q, E822K, E826K) constructs. Login to comment
99 Oocytes expressing the mutant R-domain protein (H620Q), in which a predominantly positively charged histidine was substituted by a less charged glutamine (at pH 7.2), showed a much larger conductance activated by application of phos-cock. Login to comment
102 The conductance activated by phos-cock in H620Q expressed cells was 27.84 &#fe; 5.7 WS (n = 6). Login to comment
132 We found that the open probability of the E822K and E826K mutants was signi'cantly lower than that of wild type CFTR, whereas that of the H620Q mutant was strongly enhanced compared to wild type (not shown). Login to comment
143 A comparison of the three R-domain mutants leads to a remarkable conclusion: both mutations, E822K and E826K, in which negatively charged glutamic acids were replaced by positively charged lysine had a signi'cantly reduced CFTR channel activity, whereas the H620Q mutation, in which positively charged histidine was replaced by the more neutral amino acid glutamine, had a much higher channel activity. Login to comment

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

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

Comments [show]

None has been submitted yet.

Sentences [show]
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
76 Critical information was provided by Thilo D&#f6;rk (H620Q); R. Moss (R117H & G551D homozygotes); David Kessler, Theresa Grebe and Elizabeth Perkett (D648V); Monica Brooks and contributors to the Cystic Fibrosis Foundation Registry (G178R and G1244E); Aleksey Savov and Luba Kalaydjieva (R1070Q); and Christiane De Boeck and Harry Cuppens (G970R). Login to comment

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

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

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
2023 Interestingly, the authors demonstrated that G178R and H620Q, two CF-PI mutations that disrupt cellular HCO3 - transport, were without effect on CFTR-mediated HCO3 - transport, arguing that CF mutations influence HCO3 --transport indirectly. Login to comment

[hide] Enhancing the Potency of F508del Correction: A Mul... J Pharmacol Clin Toxicol. 2013 Aug 28;1(1):1007. Kirby EF, Heard AS, Wang XR
Enhancing the Potency of F508del Correction: A Multi-Layer Combinational Approach to Drug Discovery for Cystic Fibrosis.
J Pharmacol Clin Toxicol. 2013 Aug 28;1(1):1007., [PMID:24855632]

Abstract [show]
With better understanding of the cellular and molecular pathophysiology underlying cystic fibrosis (CF), novel drugs are being developed that specifically target the molecular defects of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel on the plasma membrane that causes CF. Starting with cell-based high-throughput screening, small molecules have been identified that are able to fix specific molecular defects of various disease-causing CFTR mutants. With the successful development of ivacaftor, a "potentiator" that enhances CFTR chloride channel activity, new types of small-molecule compounds that "correct" the misfolding and misprocessing of the most common CF-causing mutation, F508del, are actively being sought for. Recent studies focused on the potential mechanisms of action of some of the investigational CFTR "correctors" shed new light on how the F508del mutant can be targeted in an attempt to ameliorate the clinical symptoms associated with CF. A multi-layer combinational approach has been proposed to achieve the high-potency correction necessary for significant clinical outcome. The mechanistic insights obtained from such studies will shape the future therapeutics development for the vast majority of CF patients.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
84 H620Q substitution at the C-terminal region of NBD1 is known to increase the channel open probability of CFTR [34]. Login to comment
86 NMR studies of human F508del NBD1 either carrying the H620Q mutation or treated with CFFT-001 revealed a similar disruption of the interactions between the (b2;-strands S3, S9, and S10 and the C-terminal helices H8 and H9, suggesting a similar conformational shift [35]. Login to comment
87 Differential scanning calorimetry showed a reduced Tm of NBD1 Kirby et al. Page 5 in the presence of H620Q or CFFT-001, suggesting that CFFT-001 binds to a less stable conformation of NBD1 [35]. Login to comment