ABCMdb

ABCC7 p.Val510Ala

Predicted by SNAP2:	A: N (82%), C: N (72%), D: N (57%), E: N (72%), F: N (72%), G: N (72%), H: N (66%), I: N (93%), K: N (82%), L: N (87%), M: N (87%), N: N (72%), P: N (66%), Q: N (82%), R: N (78%), S: N (72%), T: N (87%), W: D (66%), Y: N (78%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, W: N, Y: N,

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[hide] Correctors promote maturation of cystic fibrosis t... J Biol Chem. 2007 Nov 16;282(46):33247-51. Epub 2007 Oct 2. Wang Y, Loo TW, Bartlett MC, Clarke DM
Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein.
J Biol Chem. 2007 Nov 16;282(46):33247-51. Epub 2007 Oct 2., 2007-11-16 [PMID:17911111]

Abstract [show]
The most common cause of cystic fibrosis (CF) is defective folding of a cystic fibrosis transmembrane conductance regulator (CFTR) mutant lacking Phe(508) (DeltaF508). The DeltaF508 protein appears to be trapped in a prefolded state with incomplete packing of the transmembrane (TM) segments, a defect that can be repaired by expression in the presence of correctors such as corr-4a, VRT-325, and VRT-532. To determine whether the mechanism of correctors involves direct interactions with CFTR, our approach was to test whether correctors blocked disulfide cross-linking between cysteines introduced into the two halves of a Cys-less CFTR. Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 degrees C, we found that maturation could be restored if Val(510) was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. The V510D mutation also promoted maturation of DeltaF508 CFTR. The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Correctors and the CFTR channel blocker benzbromarone, but not P-glycoprotein substrates, inhibited cross-linking of mutant I340C(TM6)/S877C(TM7). These results suggest that corrector molecules such as corr-4a interact directly with CFTR.

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No. Sentence Comment
3 Although replacement of the 18 endogenous cysteines of CFTR with Ser or Ala yields a Cys-less mutant that does not mature at 37 °C, we found that maturation could be restored if Val510 was changed to Ala, Cys, Ser, Thr, Gly, Ala, or Asp. Login to comment
5 The Cys-less/V510A mutant was used for subsequent cross-linking analysis as it yielded relatively high levels of mature protein that was functional in iodide efflux assays. Login to comment
6 We tested for cross-linking between cysteines introduced into TM6 and TM7 of Cys-less CFTR/V510A because cross-linking between TM6 and TM7 of P-glycoprotein, the sister protein of CFTR, was inhibited with the corrector VRT-325. Login to comment
7 Cys-less CFTR/V510A mutant containing cysteines at I340C(TM6) and S877C(TM7) could be cross-linked with a homobifunctional cross-linker. Login to comment
26 Mutations I340C(TM6) and S877C(TM7) were inserted into Cys-less CFTR/V510A singly or together. Login to comment
28 The ⌬F508 mutation was also introduced into the Cys-less/V510A and I340C(TM6)/S877C(TM7)/Cys-less/ V510A mutants. Login to comment
46 Disulfide Cross-linking Analysis-CFTR mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR was expressed in HEK 293 cells. Login to comment
68 The mutants were expressed in HEK 293 cells at 37 °C, and whole cell extracts were subjected to immunoblot analysis. Fig. 2B shows that changing Val510 to Thr, Gly, Ala, Ser, or Asp promoted maturation of Cys-less CFTR. Login to comment
70 Stable BHK cell lines expressing mutants V510A, V510C, V510S, V510G, or V510D were generated for use in iodide efflux assays. Login to comment
72 The results of the most active (V510A) and least active (V510D) mutants are shown in Fig. 2C. Login to comment
82 ACCELERATED PUBLICATION: CFTR TM Domain Cross-linking 33248 To test whether the Val510 changes could also promote maturation of ⌬F508 CFTR (in a wild-type background), we introduced the Val510 mutations that promoted maturation of Cys-less CFTR (Val510 to Cys, Gly, Ala, Ser, Asp, or Thr) into ⌬F508 CFTR. Login to comment
84 The Cys-less/V510A CFTR mutant was then used for disulfide cross-linking analysis to test whether correctors directly interacted with the protein. Since corrector VRT-325 inhibited cross-linking of P-gp mutant L339C(TM6)/F728C(TM7), we aligned TM segments (6 and 7) of P-gp and CFTR to identify the equivalent residues in CFTR. Login to comment
87 The 16 double cysteine mutants were initially constructed in a Cys-less CFTR lacking V510A, so they required expression at 27 °C for maturation of CFTR (data not shown). Login to comment
90 The I340C(TM6)/S877C(TM7) mutations were then introduced into the Cys-less/V510A CFTR background. Login to comment
91 The I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR mutant was expressed in HEK 293 cells, and samples were treated with methanethiosulfonate cross-linkers of various sizes and subjected to immunoblot analysis. Login to comment
93 Fig. 3A shows that mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR could be cross-linked with various methanethiosulfonate cross-linkers. Login to comment
98 C, iodide efflux assays were performed on stable BHK cell lines expressing Cys-less/V510A CFTR (open circles) or Cys-less/V510D CFTR (closed squares). Login to comment
103 Disulfide cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
104 A, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were treated with (ϩ) or without (-) 0.1 mM of the indicated cross-linkers for 16 min at 22 °C. Login to comment
108 B, immunoblot analysis of HEK 293 cells expressing Cys-less/V510A CFTR, mutants I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR, I340C(TM6) Cys-less/V510A CFTR, or S877C(TM7) Cys-less/V510A CFTR were treated with (ϩ) or without (-) cross-linker M11M. Login to comment
113 The I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR mutant was used as a reporter molecule to study CFTR interactions with CFTR correctors such as corr-4a (4), VRT-325 (5, 17), and VRT-532 (5, 6). Login to comment
114 We first tested the system by using a compound that binds within the CFTR channel pore (at the interface between the two TMDs) and to see whether it could block cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
116 Accordingly, HEK 293 cells expressing mutant I340C(TM6)/ S877C(TM7) Cys-less/V510A CFTR were preincubated with various concentrations of benzbromarone and then treated with M11M cross-linker. Login to comment
120 Verapamil (Fig. 4E) and demecolcine (Fig. 4F) did not inhibit cross-linking of mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
122 We could not test whether correctors blocked cross-linking in the ⌬F508/ I340C(TM6)/S877C(TM7)/Cys-less/V510A mutant because mature CFTR was not detected in mutants ⌬F508/Cys-less/ V510A or ⌬F508/I340C(TM6)/S877C(TM7)/Cys-less/V510A even when expressed at 27 °C (Fig. 4, G and H). Login to comment
131 Although V510D was the most efficient suppressor mutation because it promoted maturation of both Cys-less and ⌬F508 CFTRs, it was less useful than the V510A change in Cys-less CFTR because it showed reduced iodide efflux activity. Login to comment
133 The ability of VRT-325, VRT-532, and corr-4a (Fig. 4) to block cross-linking of I340C(TM6)/S877C(TM7) Cys-less/ V510A CFTR suggests that they interact directly with CFTR. Login to comment
138 Effect of CFTR modulators and correctors on disulfide cross-linking of CFTR mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR. Login to comment
139 HEK 293 cells expressing mutant I340C(TM6)/S877C(TM7) Cys-less/V510A CFTR were incubated at 22 °C for 30 min in the absence (None) or presence of theindicatedcompounds.Thesampleswerethentreatedwith(ϩ)orwithout (-) 0.1 mM M11M at 22 °C for 16 min. Login to comment
141 Immunoblot analysis was also performed on mutants Cys-less/V510A (G) or I340C(TM6)/S877C(TM7)/Cys-less/V510A (H) with (⌬F508) or without (-) the ⌬F508 mutation that were transiently expressed in HEK 293 cells for 24 h at 27 °C. Login to comment

[hide] Correctors promote folding of the CFTR in the endo... Biochem J. 2008 Jul 1;413(1):29-36. Loo TW, Bartlett MC, Clarke DM
Correctors promote folding of the CFTR in the endoplasmic reticulum.
Biochem J. 2008 Jul 1;413(1):29-36., 2008-07-01 [PMID:18361776]

Abstract [show]
Cystic fibrosis (CF) is most commonly caused by deletion of a residue (DeltaF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) protein. The misfolded mutant protein is retained in the ER (endoplasmic reticulum) and is not trafficked to the cell surface (misprocessed mutant). Corrector molecules such as corr-2b or corr-4a are small molecules that increase the amount of functional CFTR at the cell surface. Correctors may function by stabilizing CFTR at the cell surface or by promoting folding in the ER. To test whether correctors promoted folding of CFTR in the ER, we constructed double-cysteine CFTR mutants that would be retained in the ER and only undergo cross-linking when the protein folds into a native structure. The mature form, but not the immature forms, of M348C(TM6)/T1142C(TM12) (where TM is transmembrane segment), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutants were efficiently cross-linked. Mutations to the COPII (coatamer protein II) exit motif (Y(563)KDAD(567)) were then made in the cross-linkable cysteine mutants to prevent the mutant proteins from leaving the ER. Membranes were prepared from the mutants expressed in the absence or presence of correctors and subjected to disulfide cross-linking analysis. The presence of correctors promoted folding of the mutants as the efficiency of cross-linking increased from approx. 2-5% to 22-35%. The results suggest that correctors interact with CFTR in the ER to promote folding of the protein into a native structure.

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No. Sentence Comment
40 The location of the V510A and cross-linkable cysteine residues [(M348C(TM6)/T1142C(TM12) (dashed line), T351C(TM6)/T1142C(TM12) (solid line) and W356C(TM6)/W1145C(TM12) (dotted line) mutants] are indicated. Login to comment
47 The cysteine-less CFTR also contained a V510A mutation (cysteine-less/V510A) that promoted maturation at 37◦ C [17]. Login to comment
48 The double-cysteine mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/ T1142C(TM12) and W356C(TM6)/W1145C(TM12) that were shown to be cross-linkable with M8M (3,6-dioxaoctane-1,8-bismethanethiosulfonate) cross-linker [3] were introduced into the cysteine-less/V510A CFTR. Login to comment
93 The cysteine-less/ V510A CFTR mutant exhibited channel activity at the cell surface [17]. Login to comment
95 To test whether the mature and immature forms of cysteine-less CFTR/V510A still exhibited structural differences, the Figure 2 Disulfide cross-linking of cysteine mutants in wild-type or cysteine-less/V510A CFTR background Wild-type CFTR containing the M348C(TM6)/T1142C(TM12) mutations (A) or cysteine-less/V510A CFTR containing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutation (B) were expressed in HEK-293 cells. Login to comment
99 M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations were introduced into cysteine-less/V510A CFTR. Login to comment
103 There was a little aggregation of immature CFTR, however, when cysteine-less/V510A containing the M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/ W1145C(TM12) mutations were treated with M8M cross-linker (Figure 2B). Login to comment
105 In contrast, no cross-linked product was detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142(TM12) and W1145C(TM12) in the cysteine-less/V510A background were each treated with M8M cross-linker (results not shown). Login to comment
106 These results suggest that the M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C(TM12) mutations in the cysteine-less/V510A CFTR background could act as reporters for monitoring folding of the TMDs because only mature CFTR shows cross-linking. Login to comment
112 The reactions were Figure 3 Concentration-dependence of M8M cross-linking of cysteine mutants (A) HEK-293 cells expressing M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background were suspended in PBS. Login to comment
124 Slow-migrating product was not detected when single-cysteine mutants M348C(TM6), T351C(TM6), W356C(TM6), T1142C(TM12) and W1145(TM12) in cysteine-less CFTR/V510A were each treated with M8M (results not shown) We then examined whether correctors, channel blockers or potentiators inhibited cross-linking of M348C(TM6)/T1142C- (TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/ W1145C(TM12) mutants. Login to comment
148 (C) M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A CFTR background was expressed in the absence (-) or presence (+) of 15 μM corr-4a. Login to comment
151 (D) Membranes were prepared from HEK-293 cells expressing M348C(TM6)/T1142C(TM12)/Y563N, T351C(TM6)/T1142C(TM12)/Y563N or W356C(TM6)/W1145C(TM12)/Y563N mutant in the cysteine-less/V510A background that were grown in the absence (-) or presence (+) 15 μM corr-4a. Login to comment
166 The Y563N mutation was then introduced into M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant in the cysteine-less/V510A background. Login to comment
170 These results indicate that the COPII exit motif mutations in cysteine-less/V510A CFTR still blocked maturation even when expressed in the presence of a corrector. Login to comment
171 Membranes were then prepared from cells transfected with M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) or W356C(TM6)/W1145C(TM12) mutant cDNA in the cysteine-less/V510A/Y563N background and grown in the presence or absence of corr-4a. Login to comment
178 To confirm that the corrector was modulating folding in the ER, we expressed wild-type CFTR and T351C(TM6)/T1142C- (TM12)/Y563N/cysteine-less/V510A mutant in the absence or presence of brefeldin A before cross-linking with M8M cross-linker. Login to comment
182 Cross-linking of T351C(TM6)/ T1142C(TM12)/Y563N/cysteine-less/V510A mutant expressed in the presence of brefeldin A with or without corr-4a showed that there was more cross-linked product when corr-4a was present (Figure 6B). Login to comment
186 To test whether corr-4a promoted folding of the F508 mutant in the ER, the F508 mutation was introduced into Figure 6 Effect of brefeldin A on maturation of wild-type CFTR and cross-linking analysis of T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant HEK-293 cells were transfected with wild-type or T351C(TM6)/T1142C(TM12)/Y563N/cysteineless/V510A mutant CFTR cDNAs. Login to comment
189 (B) After 16 h, the medium in cells expressing T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A mutant was replaced with fresh medium containing 10 μg/ml brefeldin A with (+) or without (-) 15 μM corr-4a. Login to comment
193 the double-cysteine mutants M348C(TM6)/T1142C-(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12) in the Y563N/cysteine-less/V510A CFTR background. Login to comment
196 Expression in the presence of corr-4a, however, increased the yield of cross-linked product of F508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/ V510A, F508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510Aand F508/W356C(TM6)/W1145C(TM12)/Y563N/ cysteine-less/V510A mutants to 5, 11 and 10% respectively (Figure 7). Login to comment
207 The presence Figure 7 Effects of F508 mutation on cross-linking of COPII cysteine mutants HEK-293 cells expressing CFTR F508/M348C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A, F508/T351C(TM6)/T1142C(TM12)/Y563N/cysteine-less/V510A or F508/ W356C(TM6)/W1145C(TM12)/Y563N/cysteine-less/V510A mutant were grown in the absence (-) or presence (+) of 15 μM corr-4a. Membranes were prepared, and cross-linking with M8M cross-linker was performed. Login to comment

[hide] Processing mutations disrupt interactions between ... J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16. Loo TW, Bartlett MC, Clarke DM
Processing mutations disrupt interactions between the nucleotide binding and transmembrane domains of P-glycoprotein and the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2008 Oct 17;283(42):28190-7. Epub 2008 Aug 16., 2008-10-17 [PMID:18708637]

Abstract [show]
P-glycoprotein (P-gp, ABCB1) is an ATP-dependent drug pump. Each of its two homologous halves contains a transmembrane domain (TMD) that has six transmembrane (TM) segments and a nucleotide-binding domain (NBD). Determining how the two halves interact may provide insight into the folding of P-gp as the drug-binding pocket and nucleotide-binding sites are predicted to be at the interface between the two halves. Here, we present evidence for NBD1-TMD2 and NBD2-TMD1 interactions. We also show that TMD-NBD interactions in immature and mature P-gp can be affected by the presence of a processing mutation. We found that the NBD-TMD mutants L443C(NBD1)/S909C(TMD2) and A266C(TMD1)/F1086C(NBD2) could be cross-linked at 0 degrees C with oxidant (copper phenanthroline). Cross-linking was inhibited by vanadate-trapping of nucleotide. The presence of a processing mutation (G268V/L443C(NBD1)/S909C(TMD2); L1260A/A266C(TMD1)/F1086C(NBD2)) resulted in the synthesis of the immature (150 kDa) protein as the major product and the mutants could not be cross-linked with copper phenanthroline. Expression of the processing mutants in the presence of a pharmacological chaperone (cyclosporin A), however, resulted in the expression of mature (170 kDa) protein at the cell surface that could be cross-linked. Similarly, CFTR mutants A274C(TMD1)/L1260C(NBD2) and V510C(NBD1)/A1067C(TMD2) could be cross-linked at 0 degrees C with copper phenanthroline. Introduction of DeltaF508 mutation in these mutants, however, resulted in the synthesis of immature CFTR that could not be cross-linked. These results suggest that establishment of NBD interactions with the opposite TMD is a key step in folding of ABC transporters.

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No. Sentence Comment
51 We also constructed the V510C(NBD1)/A1067C(TMD2) CFTR mutant because position V510C or V510A promoted maturation of Cys-less CFTR (22). Login to comment
179 It was previously shown that mutations V510C or V510A promoted maturation of Cys-less CFTR (22). Login to comment

[hide] Cysteine-independent inhibition of the CFTR chlori... Br J Pharmacol. 2009 Jul;157(6):1065-71. Epub 2009 May 19. Li MS, Demsey AF, Qi J, Linsdell P
Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate.
Br J Pharmacol. 2009 Jul;157(6):1065-71. Epub 2009 May 19., [PMID:19466983]

Abstract [show]
BACKGROUND AND PURPOSE: Methanethiosulphonate (MTS) reagents are used extensively to modify covalently cysteine side chains in ion channel structure-function studies. We have investigated the interaction between a widely used negatively charged MTS reagent, (2-sulphonatoethyl) methanethiosulphonate (MTSES), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. EXPERIMENTAL APPROACH: Patch clamp recordings were used to study a 'cys-less' variant of human CFTR, in which all 18 endogenous cysteine residues have been removed by mutagenesis, expressed in mammalian cell lines. Use of excised inside-out membrane patches allowed MTS reagents to be applied to the cytoplasmic face of active channels. KEY RESULTS: Intracellular application of MTSES, but not the positively charged MTSET, inhibited the function of cys-less CFTR. Inhibition was voltage dependent, with a K(d) of 1.97 mmol x L(-1) at -80 mV increasing to 36 mmol x L(-1) at +80 mV. Inhibition was completely reversed on washout of MTSES, inconsistent with covalent modification of the channel protein. At the single channel level, MTSES caused a concentration-dependent reduction in unitary current amplitude. This inhibition was strengthened when extracellular Cl(-) concentration was decreased. CONCLUSIONS AND IMPLICATIONS: Our results indicate that MTSES inhibits the function of CFTR in a manner that is independent of its ability to modify cysteine residues covalently. Instead, we suggest that MTSES functions as an open channel blocker that enters the CFTR channel pore from its cytoplasmic end to physically occlude Cl(-) permeation. Given the very widespread use of MTS reagents in functional studies, our findings offer a broadly applicable caveat to the interpretation of results obtained from such studies.

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28 To facilitate channel protein maturation and expression in the cell membrane, the V510A mutation (Wang et al., 2007) was introduced into this construct using the QuikChange site directed mutagenesis system as described previously (Gong et al., 2002) and verified by DNA sequencing. Login to comment
29 Baby hamster kidney (BHK) and human embryonic kidney cells were transiently transfected with pIRES2-EGFP-cys-less/V510A CFTR cDNA as described previously (Gong et al., 2002), except that 24 h after transfection, cells were transferred to 27°C to promote mature protein expression (see Supporting Information, Figure S1). Login to comment
32 Once trafficked to the cell membrane (see Supporting Information, Figure S1), the properties of cys-less/V510A CFTR channel currents appeared identical to those of wild-type CFTR studied previously, with the exception that single channel conductance appeared increased (see Figures 2 and 3). Login to comment
55 Results Expression of cys-less/V510A CFTR in BHK cells led to the appearance of PKAand ATP-dependent PPi-stimulated macroscopic currents when the cells were grown at 27°C. Examples of such currents, which had similar properties to those of wild-type CFTR studied many times previously (e.g. Linsdell and Hanrahan, 1998; Gong et al., 2002), are shown in Figure 1. Login to comment
66 (A) An example of the leak-subtracted macroscopic current-voltage relationship for cys-less/V510A-CFTR following maximal current stimulation with protein kinase A catalytic subunit (PKA), adenosine 5'-triphosphate (ATP) and pyrophosphate (PPi). Login to comment

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

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

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214 We also introduced the V510A substitution into the cysteine-free mutants, which is known to promote their maturation (35). Login to comment

[hide] Regulation of conductance by the number of fixed p... J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8. Zhou JJ, Li MS, Qi J, Linsdell P
Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.
J Gen Physiol. 2010 Mar;135(3):229-45. Epub 2010 Feb 8., [PMID:20142516]

Abstract [show]
Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.

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No. Sentence Comment
27 Cys-less CFTR also included a mutation in the first nucleotide-binding domain (V510A) to increase protein expression in the cell membrane (Li et al., 2009). Login to comment

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

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

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30 Subsequently, an additional V510A mutation was introduced to promote expression (Wang et al., 2007). Login to comment
31 A single cysteine was then engineered into each position in TM6 on the cysless/V510A background using the QuikChange XL kit (Agilent Technologies). Login to comment
33 Because all of our studies were performed under this cysless/ V510A background, we will refer to this construct as our wild-type (WT) cysless control. Login to comment

[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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64 To improve expression of a Cys-free CFTR-based construct, V510A was inserted (31), and cells expressing the construct were grown for 1-2 days at 24 °C and then for 2-5 h at 37 °C before measurements. Login to comment
147 Fig. 5A shows that internal diamide (10 ␮M) suppressed ϳ30% of channel activity of a H950C/S832C/ V510A construct, and suppression was partially reversed by FIGURE 2. Login to comment
227 A-E, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing mutants H950C/S832C/V510A (A), H950C/V510A (B), S832C/V510A (C), H954C (D), and the WT hCFTR construct (E). Login to comment

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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69 To improve expression, cells expressing Cys-free CFTR-based constructs inserted with V510A (32) were grown for 1-2 days at 24 °C and then for 2-5 h at 37 °C before measurements. Login to comment

[hide] Repair of CFTR folding defects with correctors tha... Methods Mol Biol. 2011;741:23-37. Loo TW, Clarke DM
Repair of CFTR folding defects with correctors that function as pharmacological chaperones.
Methods Mol Biol. 2011;741:23-37., [PMID:21594776]

Abstract [show]
The major cause of cystic fibrosis is the presence of processing mutations in CFTR (such as deletion of Phe-508 (F508del-CFTR)) that disrupt folding of the protein and trafficking to the cell surface. Processing mutations appear to inhibit folding of CFTR so that it accumulates in the endoplasmic reticulum as a partially folded protein. Expressing the proteins in the presence of small molecules called correctors can repair CFTR folding defects. Some correctors appear to function as pharmacological chaperones that specifically bind to the CFTR processing mutants and induce them to complete the folding process. In this chapter, we describe techniques to examine the effects of correctors on folding of CFTR processing mutants.

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247 To improve the maturation efficiency of Cys-less CFTR, cysteines 590 and 592 were replaced with leucines (16) and valine 510 was changed to alanine (17). Login to comment

[hide] Functional arrangement of the 12th transmembrane r... Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28. Qian F, El Hiani Y, Linsdell P
Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant.
Pflugers Arch. 2011 Oct;462(4):559-71. Epub 2011 Jul 28., [PMID:21796338]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged into two pseudo-symmetrical groups of six. While TM6 in the N-terminal TMs is known to line the pore and to make an important contribution to channel properties, much less is known about its C-terminal counterpart, TM12. We have used patch clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of TM12 in a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM12 residues N1138, M1140, S1141, T1142, Q1144, W1145, V1147, N1148, and S1149 when applied to the cytoplasmic side of open channels. Cysteines sensitive to internal MTS reagents were not modified by extracellular [2-(trimethylammonium)ethyl] MTS, consistent with MTS reagent impermeability. Both S1141C and T1142C could be modified by intracellular [2-sulfonatoethyl] MTS prior to channel activation; however, N1138C and M1140C, located deeper into the pore from its cytoplasmic end, were modified only after channel activation. Comparison of these results with previous work on CFTR-TM6 allows us to develop a model of the relative positions, functional contributions, and alignment of these two important TMs lining the CFTR pore. We also propose a mechanism by which these seemingly structurally symmetrical TMs make asymmetric contributions to the functional properties of the channel pore.

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34 As in our recent study on CFTR-TM6 [9], we have used a human CFTR variant ("cys-less" CFTR) in which all endogenous cysteine residues had been substituted by other amino acids [32] and which also includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane [19]. Login to comment

[hide] Functional Differences in Pore Properties Between ... J Membr Biol. 2011 Oct;243(1-3):15-23. Epub 2011 Jul 28. Holstead RG, Li MS, Linsdell P
Functional Differences in Pore Properties Between Wild-Type and Cysteine-Less Forms of the CFTR Chloride Channel.
J Membr Biol. 2011 Oct;243(1-3):15-23. Epub 2011 Jul 28., [PMID:21796426]

Abstract [show]
Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.

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26 Cys-less CFTR also included a mutation in the first NBD (V510A) to increase protein expression in the cell membrane (Li et al. 2009). Login to comment
27 The V510A mutation itself is not expected to affect single-channel conductance, which has previously been reported to be similarly increased in cys-less CFTR without (Cui et al. 2006; Mense et al. 2006) and with (Li et al. 2009; Bai et al. 2010) the V510A mutation. Login to comment

[hide] Regulation of Activation and Processing of the Cys... J Biol Chem. 2012 Oct 11. Wang G, Duan DD
Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3.
J Biol Chem. 2012 Oct 11., [PMID:23060444]

Abstract [show]
NEG2, a short C-terminal segment (817-838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, K946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of D835, D836 or E838 of NEG2 to prevent channel activation by PKA. R764 or R766 of the S768 phosphorylation site of the R domain is proposed to promote channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A and E838A but also K946A reduced the PKA dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. On the other hand, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity while D835R/D836R/E838R/K946D/H950D was misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing and S768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2 and the S768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.

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124 Fig. 7 demonstrates that 10M diamide greatly suppressed the channel activity of S768C/S832C/V510A mutant but failed to affect the channel activities of both S768C/V510A and S832C/V510A. Login to comment
154 Finally, D979A in CF patients causes misprocessing (33). Login to comment
195 Effects of diamide on CFTR mutants at the R-CL3 interface. A-C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing CFTR mutants S768C/V510A (A), S832C/V510A (B), and S768C/S832C/V510A (C) by using a ramp protocol (afe;80 mV). Login to comment

[hide] Alternating access to the transmembrane domain of ... J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1. Wang W, Linsdell P
Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).
J Biol Chem. 2012 Mar 23;287(13):10156-65. Epub 2012 Feb 1., [PMID:22303012]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

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47 19), and which includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane (20). Login to comment
49 19), and which includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane (20). Login to comment

[hide] Conformational change opening the CFTR chloride ch... Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2. Wang W, Linsdell P
Conformational change opening the CFTR chloride channel pore coupled to ATP-dependent gating.
Biochim Biophys Acta. 2012 Mar;1818(3):851-60. Epub 2012 Jan 2., [PMID:22234285]

Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are controlled by ATP binding and hydrolysis by its nucleotide binding domains (NBDs). This is presumed to control opening of a single "gate" within the permeation pathway, however, the location of such a gate has not been described. We used patch clamp recording to monitor access of cytosolic cysteine reactive reagents to cysteines introduced into different transmembrane (TM) regions in a cysteine-less form of CFTR. The rate of modification of Q98C (TM1) and I344C (TM6) by both [2-sulfonatoethyl] methanethiosulfonate (MTSES) and permeant Au(CN)(2)(-) ions was reduced when ATP concentration was reduced from 1mM to 10muM, and modification by MTSES was accelerated when 2mM pyrophosphate was applied to prevent channel closure. Modification of K95C (TM1) and V345C (TM6) was not affected by these manoeuvres. We also manipulated gating by introducing the mutations K464A (in NBD1) and E1371Q (in NBD2). The rate of modification of Q98C and I344C by both MTSES and Au(CN)(2)(-) was decreased by K464A and increased by E1371Q, whereas modification of K95C and V345C was not affected. These results suggest that access from the cytoplasm to K95 and V345 is similar in open and closed channels. In contrast, modifying ATP-dependent channel gating alters access to Q98 and I344, located further into the pore. We propose that ATP-dependent gating of CFTR is associated with the opening and closing of a gate within the permeation pathway at the level of these pore-lining amino acids.

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58 The cys-less version used in the present study also includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane [19]. Login to comment

[hide] Alignment of transmembrane regions in the cystic f... J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11. Wang W, El Hiani Y, Linsdell P
Alignment of transmembrane regions in the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Gen Physiol. 2011 Aug;138(2):165-78. Epub 2011 Jul 11., [PMID:21746847]

Abstract [show]
Different transmembrane (TM) alpha helices are known to line the pore of the cystic fibrosis TM conductance regulator (CFTR) Cl(-) channel. However, the relative alignment of these TMs in the three-dimensional structure of the pore is not known. We have used patch-clamp recording to investigate the accessibility of cytoplasmically applied cysteine-reactive reagents to cysteines introduced along the length of the pore-lining first TM (TM1) of a cysteine-less variant of CFTR. We find that methanethiosulfonate (MTS) reagents irreversibly modify cysteines substituted for TM1 residues K95, Q98, P99, and L102 when applied to the cytoplasmic side of open channels. Residues closer to the intracellular end of TM1 (Y84-T94) were not apparently modified by MTS reagents, suggesting that this part of TM1 does not line the pore. None of the internal MTS reagent-reactive cysteines was modified by extracellular [2-(trimethylammonium)ethyl] MTS. Only K95C, closest to the putative intracellular end of TM1, was apparently modified by intracellular [2-sulfonatoethyl] MTS before channel activation. Comparison of these results with recent work on CFTR-TM6 suggests a relative alignment of these two important TMs along the axis of the pore. This alignment was tested experimentally by formation of disulfide bridges between pairs of cysteines introduced into these two TMs. Currents carried by the double mutants K95C/I344C and Q98C/I344C, but not by the corresponding single-site mutants, were inhibited by the oxidizing agent copper(II)-o-phenanthroline. This inhibition was irreversible on washing but could be reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between the introduced cysteine side chains. These results allow us to develop a model of the relative positions, functional contributions, and alignment of two important TMs lining the CFTR pore. Such functional information is necessary to understand and interpret the three-dimensional structure of the pore.

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60 As in our recent study on CFTR-TM6 (El Hiani and Linsdell, 2010), we have used a human CFTR variant in which all 18 endogenous cysteine residues had been substituted by other amino acids (as described in Mense et al., 2006), and which also includes a mutation in the first nucleotide-binding domain (V510A) to increase protein cross-linked residues in a current homology model of the CFTR membrane-spanning domain. Login to comment
64 Using site-directed mutagenesis, we substituted cysteines for each of 21 consecutive amino acids, from Y84 near the putative intracellular end of TM1 to R104 near the extracellular end, in a V510A cys-less background (see Materials and methods). Login to comment

[hide] Two salt bridges differentially contribute to the ... J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24. Cui G, Freeman CS, Knotts T, Prince CZ, Kuang C, McCarty NA
Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function.
J Biol Chem. 2013 Jul 12;288(28):20758-67. doi: 10.1074/jbc.M113.476226. Epub 2013 May 24., [PMID:23709221]

Abstract [show]
Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

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150 MTS Reagents Exhibit No Effects on Single Channel Amplitude of WT-CFTR and Cys-less V510A-CFTR-Because the Arg352 -Asp993 interaction appeared to make the largest contribution to stabilizing the f state, we asked whether forcing these residues to interact would lead to channels that were latched into the open state. Login to comment
155 We further added a secondary mutation to generate Cys-less V510A-CFTR in order to improve expression (9, 25). Login to comment
156 Because it was reported that Cys-less CFTR showed channel behavior similar to that of WT-CFTR with a few nominal differences (9, 26-28), we first used mutants generated on the WT-CFTR background and then confirmed the results in mutants generated on the Cys-less V510A-CFTR background. Login to comment
157 Representative data indicating that charged monofunctional MTS reagents modified the activity of WT-CFTR or Cys-less V510A-CFTR when applied cytoplasmically in excised, inside-out patches are shown in supplemental Fig. 1. Login to comment
160 Supplemental Fig. 1B shows Cys-less V510A-CFTR in the absence and presence of MTS reagents; not surprisingly, MTS reagents exhibited no effects on either single channel amplitude or NPo in the Cys-less channel. Login to comment
161 Based on these results, we resolved to use both WT-CFTR and Cys-less V510A-CFTR as backgrounds to test the consequences of modification of engineered cysteines. Login to comment
175 This added effect was lost on the Cys-less V510A background. Login to comment
179 We repeated the above experiments in R352C/Cys-less V510A-CFTR and D993C/ Cys-less V510A-CFTR to further rule out the possibility of any endogenous cysteines being involved in the process. Login to comment
181 These effects were removed only upon application of the reducing agent DTT and indicate that the Cys-less V510A-CFTR background was identical to the WT-CFTR background with respect to these experiments. Login to comment
185 Before applying MTS-2-MTS to the R352C/D993C-CFTR double mutant, we first tested the effects of this bifunctional linker on WT-CFTR (Fig. 6A) and Cys-less V510A-CFTR (Fig. 6B). Login to comment
186 MTS-2-MTS did not change the single channel amplitude of either channel but decreased NPo of WT-CFTR without changing NPo of Cys-less V510A-CFTR. Login to comment
201 Effects of 100 òe;M MTS-2-MTS on WT-CFTR (A) and Cys-less V510A-CFTR (B) at VM d1d; d1a;100 mV. A, MTS-2-MTS decreased NPo but had no effect on single channel amplitude of WT-CFTR. Login to comment
202 B, the cross-linker had no effect on either NPo or single channel amplitude of Cys-less V510A-CFTR. Login to comment

[hide] Relative contribution of different transmembrane s... Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20. Wang W, El Hiani Y, Rubaiy HN, Linsdell P
Relative contribution of different transmembrane segments to the CFTR chloride channel pore.
Pflugers Arch. 2014 Mar;466(3):477-90. doi: 10.1007/s00424-013-1317-x. Epub 2013 Aug 20., [PMID:23955087]

Abstract [show]
The membrane-spanning part of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel comprises 12 transmembrane (TM) alpha-helices, arranged in 2 symmetrical groups of 6. However, those TMs that line the channel pore are not completely defined. We used patch clamp recording to compare the accessibility of cysteine-reactive reagents to cysteines introduced into different TMs. Several residues in TM11 were accessible to extracellular and/or intracellular cysteine reactive reagents; however, no reactive cysteines were identified in TMs 5 or 11. Two accessible residues in TM11 (T1115C and S1118C) were found to be more readily modified from the extracellular solution in closed channels, but more readily modified from the intracellular solution in open channels, as previously reported for T338C in TM6. However, the effects of mutagenesis at S1118 (TM11) on a range of pore functional properties were relatively minor compared to the large effects of mutagenesis at T338 (TM6). Our results suggest that the CFTR pore is lined by TM11 but not by TM5 or TM7. Comparison with previous works therefore suggests that the pore is lined by TMs 1, 6, 11, and 12, suggesting that the structure of the open channel pore is asymmetric in terms of the contributions of different TMs. Although TMs 6 and 11 appear to undergo similar conformational changes during channel opening and closing, the influence of these two TMs on the functional properties of the narrowest region of the pore is clearly unequal.

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38 In this study, we have used two different human CFTR variants as backgrounds for further mutagenesis: wild type CFTR and "cys-less" CFTR in which all cysteines have been removed by mutagenesis [30] and that also includes a mutation in the first NBD (V510A) to increase protein expression in the cell membrane [20]. Login to comment

[hide] The cystic fibrosis V232D mutation inhibits CFTR m... Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9. Loo TW, Clarke DM
The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds.
Biochem Pharmacol. 2014 Mar 1;88(1):46-57. doi: 10.1016/j.bcp.2013.12.027. Epub 2014 Jan 9., [PMID:24412276]

Abstract [show]
Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Repair of CFTR mutants requires an understanding of the mechanisms of misfolding caused by processing mutations. Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Here, we performed mutational analysis to test for Asp232/Gln207 interactions in full-length CFTR. The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. We report that only Val232 mutations to charged amino acids severely blocked CFTR maturation. The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Mutation of Val232 to large nonpolar residues (Leu, Phe) had little effect. The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones.

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No. Sentence Comment
71 Cys-less CFTR was constructed in which all endogenous cysteines were changed to alanine except that Cys590 and Cys592 were changed to leucine and Val510 was changed to alanine. Login to comment

[hide] Metal bridges illuminate transmembrane domain move... J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20. El Hiani Y, Linsdell P
Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.
J Biol Chem. 2014 Oct 10;289(41):28149-59. doi: 10.1074/jbc.M114.593103. Epub 2014 Aug 20., [PMID:25143385]

Abstract [show]
Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane alpha-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.

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No. Sentence Comment
46 In this study, we have used a human CFTR variant in which all cysteines had been removed by mutagenesis (as described in Ref. 23) and that includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane (24). Login to comment

[hide] Functional Architecture of the Cytoplasmic Entranc... J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5. El Hiani Y, Linsdell P
Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.
J Biol Chem. 2015 Jun 19;290(25):15855-65. doi: 10.1074/jbc.M115.656181. Epub 2015 May 5., [PMID:25944907]

Abstract [show]
As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm.

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No. Sentence Comment
51 In this study, we used a human CFTR variant in which all cysteines had been removed by mutagenesis (as described in Ref. 21) and that includes a mutation in NBD1 (V510A) to increase protein expression in the cell membrane (22). Login to comment