ABCMdb

ABCC7 p.Trp356Cys

ClinVar:	c.1068G>A , p.Trp356* ? , not provided
CF databases:	c.1067G>C , p.Trp356Ser (CFTR1) ? ,
Predicted by SNAP2:	A: D (59%), C: N (53%), D: D (66%), E: D (63%), F: D (63%), G: D (66%), H: D (75%), I: D (71%), K: D (71%), L: D (71%), M: D (66%), N: D (66%), P: D (85%), Q: D (63%), R: D (71%), S: D (59%), T: D (63%), V: D (66%), Y: D (63%),
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, V: N, Y: N,

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[hide] The DeltaF508 mutation disrupts packing of the tra... J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21. Chen EY, Bartlett MC, Loo TW, Clarke DM
The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2004 Sep 17;279(38):39620-7. Epub 2004 Jul 21., 2004-09-17 [PMID:15272010]

Abstract [show]
The most common mutation in cystic fibrosis (deletion of Phe-508 in the first nucleotide binding domain (DeltaF508)) in the cystic fibrosis transmembrane conductance regulator (CFTR) causes retention of the mutant protein in the endoplasmic reticulum. We previously showed that the DeltaF508 mutation causes the CFTR protein to be retained in the endoplasmic reticulum in an inactive and structurally altered state. Proper packing of the transmembrane (TM) segments is critical for function because the TM segments form the chloride channel. Here we tested whether the DeltaF508 mutation altered packing of the TM segments by disulfide cross-linking analysis between TM6 and TM12 in wild-type and DeltaF508 CFTRs. These TM segments were selected because TM6 appears to line the chloride channel, and cross-linking between these TM segments has been observed in the CFTR sister protein, the multidrug resistance P-glycoprotein. We first mapped potential contact points in wild-type CFTR by cysteine mutagenesis and thiol cross-linking analysis. Disulfide cross-linking was detected in CFTR mutants M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12), and W356C(TM6)/W1145C(TM12) in a wild-type background. The disulfide cross-linking occurs intramolecularly and was reducible by dithiothreitol. Introduction of the DeltaF508 mutation into these cysteine mutants, however, abolished cross-linking. The results suggest that the DeltaF508 mutation alters interactions between the TM domains. Therefore, a potential target to correct folding defects in the DeltaF508 mutant of CFTR is to identify compounds that promote correct folding of the TM domains.

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No. Sentence Comment
56 TM6 point mutations (M348C, T351C, and W356C) were generated in the XbaI (bp 573) 3 KpnI (bp 1370) fragment; TM12 point mutations (T1142C and W1145C) were generated in the EcoRV (bp 2996) 3 EcoRI (bp 3643) fragment; the ⌬F508 mutation was generated in the KpnI (bp 1370) 3 ApaI (bp 2333) fragment. Login to comment
146 Three positive cross-linking mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C were identified (see Fig. 3B, band X) and selected for further study. Login to comment
148 Fig. 2B shows the expression of WT CFTR, the single cysteine mutants M348C, T351C, W356C, T1142C, and W1145C, and the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
150 The cross-linking patterns of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C showed differences when treated with different cross-linkers. Login to comment
156 The positive mutant, W356C/ W1145C, showed cross-linking with all three cross-linkers (Fig. 3B). Login to comment
159 Because the cross-linkable mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C also contained the 18 endogenous cysteines, it was important to test whether any of the single M348C, T351C, W356C, T1142C, or W1145C mutants showed evidence of cross-linking with endogenous cysteines. Login to comment
162 Reduced levels of the T352C and W356C mutants, however, were observed with M8M, and this may be due to aggregation. Login to comment
182 Despite the problems with aggregation, cross-linking analysis still appeared to be a useful assay because the putative cross-linked products were specific to the double cysteine mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C (Fig. 3B, band X). Login to comment
183 To ensure that band X was indeed the product of disulfide cross-linking between the introduced cysteines of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C, we added DTT after cross-linking. Login to comment
187 Each cDNA contained one of the cysteine mutations M348C, T351C, W356C, T1142C, or W1145C. Login to comment
188 It was found that co-expression of the single cysteine mutants M348C plus T1142C, T351C plus T1142C or W356C plus W1145C followed by treatment with the cross-linkers M5M, M8M, or M17M did not lead to cross-linking (formation of band X) (data not shown). Login to comment
189 This indicates that cross-linking occurs intramolecularly and not intermolecularly. To compare the inter-TMD interactions between WT and misprocessed CFTRs, the ⌬F508 mutation was introduced into the positive cross-linking double cysteine constructs M348C/ T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
191 As shown in Fig. 6A, incorporation of the ⌬F508 mutation into mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C abolished cross-linking. Login to comment
196 To test whether the lack of cross-linking in the ⌬F508 series of double cysteine mutants was due to inaccessibility of thiol-reactive cross-linkers to the ER membrane, we tested whether mutants M348C/T1142C, T351C/ T1142C, and W356C/W1145C (lacking ⌬F508 mutation) would still show cross-linking then they were located in an intracellular membrane. Login to comment
197 To block trafficking of the mutants to the cell surface, we pretreated cells expressing mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C with 10 ␮g/ml brefeldin A. Login to comment
212 As shown in Fig. 6B, brefeldin A blocked processing of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C. Login to comment
215 Because the mature form of mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C but not WT CFTR showed cross-linking, it was important to determine whether the mutants were still active. Login to comment
236 Both mutants T351C/T1142C and W356C/W1145C, however, exhibited ϳ40% reduction in activity compared with WT CFTR. Login to comment
248 Iodide efflux assays were performed on stable CHO cell lines expressing WT or one of the positive cross-linking double cysteine mutants (M348C/T1142C, T351C/ T1142C, and W356C/W1145C) as described under "Experimental Procedures." Login to comment
262 We were able to identify three mutants, M348C/T1142C, T351C/T1142C, and W356C/W1145C, that showed disulfide cross-linking in the mature WT background but not in the ⌬F508 background. Login to comment
263 Various control experiments were done to confirm that the mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C were indeed cross-linked through the introduced cysteines via the disulfide cross-linker. Login to comment
266 Finally, cross-linking was not observed when the cysteines in mutants M348C/T1142C, T351C/T1142C, and W356C/W1145C were co-expressed on separate CFTR molecules. Login to comment
268 The ability to detect cross-linked products between TMD1 and TMD2 such as observed with mutants M348C/ T1142C, T351C/T1142C, and W356C/W1145C could be particularly useful in future studies to monitor dynamic changes in the molecule associated with phosphorylation or ATP binding/ hydrolysis. Login to comment

[hide] The chemical chaperone CFcor-325 repairs folding d... Biochem J. 2006 May 1;395(3):537-42. Loo TW, Bartlett MC, Wang Y, Clarke DM
The chemical chaperone CFcor-325 repairs folding defects in the transmembrane domains of CFTR-processing mutants.
Biochem J. 2006 May 1;395(3):537-42., 2006-05-01 [PMID:16417523]

Abstract [show]
Most patients with CF (cystic fibrosis) express a CFTR [CF TM (transmembrane) conductance regulator] processing mutant that is not trafficked to the cell surface because it is retained in the endoplasmic reticulum due to altered packing of the TM segments. CL4 (cytoplasmic loop 4) connecting TMs 10 and 11 is a 'hot-spot' for CFTR processing mutations. The chemical chaperone CFcor-325 (4-cyclohexyloxy-2-{1-[4-(4-methoxy-benezenesulphonyl)piperazin-1-yl]-ethy l}-quinazoline) rescued most CL4 mutants. To test if CFcor-325 promoted correct folding of the TMDs (TM domains), we selected two of the CL4 mutants (Q1071P and H1085R) for disulphide cross-linking analysis. Pairs of cysteine residues that were cross-linked in mature wild-type CFTR were introduced into mutants Q1071P and H1085R. The cross-linking patterns of the Q1071P or H1085R double cysteine mutants rescued with CFcor-325 were similar to those observed with mature wild-type double cysteine proteins. These results show that CFcor-325 rescued CFTR mutants by repairing the folding defects in the TMDs.

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No. Sentence Comment
115 Mature mutant Q1071P/ M348C(TM6)/T1142C(TM12) protein was cross-linked with Figure 6 Disulphide cross-linking analysis of CFTR processing mutants HEK-293 cells expressing mutants Q1071P/M348C(TM6)/T1142C(TM12), Q1071P/T351C- (TM6)/T1142C(TM12) and Q1071P/W356C(TM6)/W1145C(TM12) (A), mutants H1085R/ M348C(TM6)/T1142C(TM12), H1085R/T351C(TM6)/T1142C(TM12) and H1085R/W356C- (TM6)/W1145C(TM12) (B) or wild-type, mutant Q1071P or mutant H1085R (C) were incubated for 48 h with (+) or without (-) 3 µM CFcor-325. Login to comment

[hide] Atomic model of human cystic fibrosis transmembran... Cell Mol Life Sci. 2008 Aug;65(16):2594-612. Mornon JP, Lehn P, Callebaut I
Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces.
Cell Mol Life Sci. 2008 Aug;65(16):2594-612., [PMID:18597042]

Abstract [show]
We describe herein an atomic model of the outward-facing three-dimensional structure of the membrane-spanning domains (MSDs) and nucleotide-binding domains (NBDs) of human cystic fibrosis transmembrane conductance regulator (CFTR), based on the experimental structure of the bacterial transporter Sav1866. This model, which is in agreement with previous experimental data, highlights the role of some residues located in the transmembrane passages and directly involved in substrate translocation and of some residues within the intracellular loops (ICL1-ICL4) making MSD/NBD contacts. In particular, our model reveals that D173 ICL1 and N965 ICL3 likely interact with the bound nucleotide and that an intricate H-bond network (involving especially the ICL4 R1070 and the main chain of NBD1 F508) may stabilize the interface between MSD2 and the NBD1F508 region. These observations allow new insights into the ATP-binding sites asymmetry and into the molecular consequences of the F508 deletion, which is the most common cystic fibrosis mutation.

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No. Sentence Comment
153 Interestingly, it appears that all the CFTR mutants for which disulfide cross-linking was detected (M348C in TM6 and T1142C in TM12; T351C in TM6 and T1142C in TM12; W356C in TM6 and W1145C in TM12) line the chloride channel pore and face each other (Fig. 3A). Login to comment

[hide] The V510D suppressor mutation stabilizes DeltaF508... Biochemistry. 2010 Aug 3;49(30):6352-7. Loo TW, Bartlett MC, Clarke DM
The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface.
Biochemistry. 2010 Aug 3;49(30):6352-7., 2010-08-03 [PMID:20590134]

Abstract [show]
Deletion of Phe508 (DeltaF508) in the first nucleotide-binding domain (NBD1) of CFTR causes cystic fibrosis. The mutation severely reduces the stability and folding of the protein by disrupting interactions between NBD1 and the second transmembrane domain (TMD2). We found that replacement of Val510 with acidic residues (but not neutral or positive residues) promoted maturation of DeltaF508-CFTR with V510D more efficiently than V510E. Promotion of DeltaF508-CFTR maturation did not require NBD2 as introduction of V510D into a DeltaNBD2/DeltaF508-CFTR mutant restored maturation to levels similar to that of full-length protein. The V510D mutation increased the half-life of mature DeltaF508-CFTR at the cell surface by about 5-fold to resemble the half-life of wild-type CFTR. It was also observed that introduction of the V510R/R1070D mutations into DeltaF508-CFTR also promoted maturation whereas the V510D/R1070A mutations did not. We propose that the V510D mutation in NBD1 promotes maturation and stabilizes DeltaF508-CFTR at the cell surface through formation of a salt bridge with Arg1070 in TMD2.

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No. Sentence Comment
27 HEK 293 cells were transfected with W356C/W1145C-, ΔF508/W356C/W1145C-, or ΔF508/V510D/W356C/W1145C-CFTR cDNAs, and the cells were incubated for 4 h at 37 °C. Login to comment
92 We previously showed that cysteines introduced into TM segments 6 (W356C) and 12 (W1145C) could be cross-linked in mature CFTR when whole cells were treated with bifunctional thiol cross-linkers (14, 15). Login to comment
96 Accordingly, the W356C and W1145C mutations were introduced into wild-type, ΔF508-, and ΔF508/V510D-CFTRs. Login to comment
100 Panels A and B of Figure 5 show that the half-lives of cross-linked wild-type/W356C/W1145C-, ΔF508/ W356C/W1145C-, and ΔF508/V510D/W356C/W1145C-CFTRs were about 12,3,and 14 h, respectively. Login to comment
103 It was possible that cysteines W356C and W1145C may influence the stability of the protein. Login to comment
131 HEK 293 cells expressing wild-type, ΔF508-, or ΔF508/ V510D-CFTRs containing the W356C and W1145C cysteines were treated with the thiol cross-linker BMH. 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
264 Cys-less CFTR, mutants W356C/W1145C and V232D/W356C/W1145C were expressed in HEK 293 cells in the absence or presence of 5 mM VX-809. Membranes were prepared Fig. 8. Login to comment
270 Cross-linked product was detected by SDS-PAGE followed by immunoblot analysis. Fig. 10A shows that the parent W356C/ W1145C CFTR readily matured in the absence of corrector VX-809 and was efficiently cross-linked with M8M or BMH (about 70% cross-linked product; Fig. 10B). Login to comment
271 No cross-linked product was detected in Cys-less CFTR or in mutant V232D/W356C/W1145C when expressed in the absence of corrector VX-809 (Fig. 10A and B). Login to comment
272 Expression of mutant V232D/W356C/W1145C in the presence of corrector however, promoted maturation of the protein such it could be efficiently cross-linked with either M8M or BMH (about 80-85% cross-linked product; Fig. 10B). Login to comment
293 (A) Membranes prepared from cells expressing mutants W356C/W1145C, V232D/W356C/W1145C or Cys-less CFTR in the absence (none) or presence of corrector VX-809 were treated without (none) or with cross-linkers (X-linkers) M8M or BMH for 10 min at 20 8C. Login to comment
329 (A) CFTR mutant W356C/W1145C that does not contain any processing mutation can fold into a native structure resulting in proper contacts between the various domains and packing of the TM segments such that W356C and W1145C can be cross-linked with cross-linkers M8M or BMH (orange line). Login to comment
331 Packing of the TM segments is incomplete such that W356C and W1145C cannot be cross-linked. Login to comment
332 (C) Studies [19,20] suggest that corrector VX-809 interacts with TMD1 to induce V232D to complete the folding process to yield a native structure in which W356C and W1145C can be cross-linked (orange line). Login to comment