ABCMdb

ABCC7 p.Trp1145Cys

None has been submitted yet.

Publications

PMID: 15272010 [PubMed] Chen EY et al: "The DeltaF508 mutation disrupts packing of the transmembrane segments of the cystic fibrosis transmembrane conductance regulator."

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
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

PMID: 18361776 [PubMed] Loo TW et al: "Correctors promote folding of the CFTR in the endoplasmic reticulum."

No. Sentence Comment

83 It was shown that pairs of cysteine residues introduced into TM6 and TM12 [M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12)] of CFTR (Figure 1) could be cross-linked with MTS (methanethiosulfonate) cross-linkers when the protein matured and was delivered to the cell surface. Login to comment
135 The reactions were stopped by addition of SDS sample buffer containing no reducing agent, and samples were subjected to immunoblot analysis. Figure 4 shows that the channel blockers benzbromarone and glibenclamide (structures shown in Figure 1B) almost completely inhibited cross-linking of M348C(TM6)/T1142C(TM12), T351C(TM6)/T1142C(TM12) and W356C(TM6)/W1145C- (TM12) mutants. 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

PMID: 18597042 [PubMed] Mornon JP et al: "Atomic model of human cystic fibrosis transmembrane conductance regulator: membrane-spanning domains and coupling interfaces."

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

PMID: 20590134 [PubMed] Loo TW et al: "The V510D suppressor mutation stabilizes DeltaF508-CFTR at the cell surface."

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

PMID: 21796338 [PubMed] Qian F et al: "Functional arrangement of the 12th transmembrane region in the CFTR chloride channel pore based on functional investigation of a cysteine-less CFTR variant."

No. Sentence Comment

80 Application of MTSES (200 μM) or MTSET (2 mM) to the intracellular solution after channel activation with PKA, ATP, and PPi significantly altered macroscopic current amplitude in nine out of 19 cysteine-substituted mutants tested (N1138C, M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C, and S1149C; Figs. 1 and 2). Login to comment
84 Closer to the intracellular end of TM12 (in T1142C, Q1144C, and W1145C), macroscopic current amplitude was decreased by MTSES application but increased by MTSET (Figs. 1 and 2). Login to comment

PMID: 22042986 [PubMed] Bai Y et al: "Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)."

No. Sentence Comment

64 Fig. S6 depicts a representative trace showing modification of cysless/W1145C channels in the absence of ATP. Login to comment
154 (A and B) Single-channel recordings at 50 mV show that application of MTSET+ increases unitary current amplitudes for cysless/N1148C (A) or cysless/W1145C (B). Login to comment
155 Linear fits to single-channel current measurements at different voltages yield unitary conductance values for cysless/ N1148C (A) of 7.1 pS before (black) and 9.2 pS after (blue) MTSET+ treatment, for cysless/W1145C (B) of 7.6 pS before (black) and 10.1 pS after (blue) MTSET+ treatment. Login to comment
159 (A) In an inside-out patch containing thousands of cysless/W1145C-CFTR channels, MTSES was first (indicated by orange lines) applied in the presence of 200 µM glibenclamide (yielding 77% steady-state blockade), and then (red lines) in the absence of glibenclamide after the chemical modification was reversed by the reducing reagent, DTT. Login to comment

PMID: 24412276 [PubMed] Loo TW et al: "The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds."

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

PMID: 25143385 [PubMed] El Hiani Y et al: "Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel."

No. Sentence Comment

51 To investigate potential Cd2af9; bridges formed between pore-lining cysteine side chains exposed in the inner vestibule of the CFTR pore, we combined individual cysteines that we previously found to be accessible to cytoplasmically applied methanethiosulfonate reagents in three important pore-lining TMs: TM1 (K95C, Q98C) (13), TM6 (I344C, V345C, M348C, A349C) (15), and TM12 (M1140C, S1141C, T1142C, Q1144C, W1145C, V1147C, N1148C) (16), to generate a total of 50 double cysteine mutants (8 TM1:TM6; 14 TM1:TM12; 28 TM6:TM12). Login to comment
71 In contrast, the remaining seven double cysteine mutants, namely I344C/S1141C (Fig. 2, C and D), V345C/S1141C, M348C/ S1141C (Fig. 2, C and E), M348C/V1144C, M348C/W1145C, M348C/V1147C, and M348C/N1148C, all showed increased sensitivity to Cd2af9; , leading to a significant decrease in Ki as compared with either of the single cysteine mutants from which they were derived (estimated Ki values b0d; 50 òe;M; Fig. 3). Login to comment
137 Thus, M348C is able to form Cd2af9; bridges with cysteines at multiple positions in TM12 (S1141C, Q1144C, W1145C, V1147C, N1148C) (Fig. 8B), and S1141C is able to form Cd2af9; bridges with cysteines both in TM1 (K95C) and in TM6 (I344C, V345C, M348C) (Fig. 8C). Login to comment