ABCC7 p.Thr1121Cys
| Predicted by SNAP2: | A: N (72%), C: D (66%), D: D (53%), E: D (66%), F: D (71%), G: N (57%), H: D (71%), I: D (71%), K: D (71%), L: D (53%), M: D (59%), N: N (66%), P: D (66%), Q: N (53%), R: D (63%), S: N (82%), V: D (53%), W: D (80%), Y: D (59%), |
| 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, V: N, W: D, Y: N, |
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[hide] Novel residues lining the CFTR chloride channel po... J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19. Fatehi M, Linsdell P
Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.
J Membr Biol. 2009 Apr;228(3):151-64. Epub 2009 Apr 19., [PMID:19381710]
Abstract [show]
Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.
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No. Sentence Comment
71 As described previously for modification of cysteines introduced into TM6 (Fatehi and Linsdell 2008) and the extracellular loop between TMs 1 and 2 (Zhou et al. 2008), MTSET and MTSES altered the IREL-V shape in S1118C, T1121C, T1122C, G1127C, V1129C, I1131C and I1132C.
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ABCC7 p.Thr1121Cys 19381710:71:220
status: NEW72 Of 21 cysteine mutants studied, only six significantly altered I-V relationship shape in the absence of external MTS reagents (Fig. 3a), with S1118C, T1121C, T1122C, G1127C and A1136C all causing significant inward rectification and V1129C showing outward rectification.
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ABCC7 p.Thr1121Cys 19381710:72:150
status: NEW84 Unitary currents at depolarized voltages were significantly decreased in S1118C, T1121C, T1122C and G1127C and significantly increased in V1129C (Fig. 5b).
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ABCC7 p.Thr1121Cys 19381710:84:81
status: NEW85 This resulted in changes in the shape of the i-V relationship, causing inward rectification in the case of S1118C, T1121C, T1122C and G1127C and outward rectification in the case of V1129C (Figs. 4b, 5c).
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ABCC7 p.Thr1121Cys 19381710:85:115
status: NEW91 Indeed, changes in unitary current amplitude were observed in S1118C, T1121C, T1122C, G1127C, V1129C, I1131C and I1132C, but not wild-type, when MTS reagents were included in the pipette solution (Fig. 6).
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ABCC7 p.Thr1121Cys 19381710:91:72
status: NEW118 Four mutations (S1118C, T1121C, T1122C, G1127C) led to significant decreases in unitary current amplitude (Fig. 5b), which were relatively strongly affected by MTS modification-in each case conductance was further decreased by reaction with MTSES and increased to near wild-type levels by MTSET (Fig. 9a).
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ABCC7 p.Thr1121Cys 19381710:118:24
status: NEW124 Under these conditions, SCN- block was significantly strengthened in I1132C (at hyperpolarized and depolarized voltages), S1118C (at hyperpolarized voltages), T1121C and V1129C (at depolarized voltages) and I1131C (at very depolarized voltages only) Fig. 4 Single-channel currents carried by cysteine mutant forms of CFTR.
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ABCC7 p.Thr1121Cys 19381710:124:159
status: NEW125 a Example single-channel currents carried by wild-type, S1118C, T1121C, T1122C and V1129C, at membrane potentials of ?60 (top) and -60 (bottom) mV.
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ABCC7 p.Thr1121Cys 19381710:125:64
status: NEW133 Under these conditions, SCN- permeability was significantly increased in S1118C and (to a lesser extent) T1122C and G1127C and unaltered in T1121C, V1129C, I1131C and I1132C (Fig. 11).
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ABCC7 p.Thr1121Cys 19381710:133:140
status: NEW158 Of the seven mutants that were functionally modified by MTS reagents, five (S1118C, T1121C, T1122C, G1127C, V1129C) also showed significantly altered unitary current amplitude in the absence of MTS modification (Figs. 4, 5).
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ABCC7 p.Thr1121Cys 19381710:158:84
status: NEW161 a S1118C (d), T1121C (j), T1122C (), G1127C (h); b V1129C (m), I1131C (r), I1132C (.).
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ABCC7 p.Thr1121Cys 19381710:161:14
status: NEW183 We speculate that one group of reactive mutants (S1118C, T1121C, T1122C, G1127C) is located relatively deep in the pore from the outside and that the other (V1129C, Fig. 11 Thiocyanate permeability of mutants.
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ABCC7 p.Thr1121Cys 19381710:183:57
status: NEW188 In this scenario, charge-neutral mutations deeper in the pore (S1118C, T1121C, T1122C, G1127C) (Fig. 9a) disrupt Cl- movement in the pore in a nonelectrostatic fashion, leading to reduced unitary currents at depolarized voltages, as described previously for TM6 mutations (McDonough et al. 1994; Linsdell et al. 1998; Linsdell 2001a).
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ABCC7 p.Thr1121Cys 19381710:188:71
status: NEW[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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None has been submitted yet.
No. Sentence Comment
78 MTSES did not affect T1121C currents; however, these were inhibited by CFTRinh-172 (5 bc;M; hatched bar).
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ABCC7 p.Thr1121Cys 23955087:78:21
status: NEW117 Application of MTSES (200 bc;M) following channel activation with PKA and ATP caused a decrease in macroscopic current amplitude in I1112C, T1115C and S1118C, but not in T1121C (Fig. 2a, b) or in I1109C, F1110C, F1111C, A1113C, V1114C, F1116C, or I1117C (Fig. 2c).
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ABCC7 p.Thr1121Cys 23955087:117:173
status: NEW122 Previously it was shown that S1118C, T1121C, and T1122C-but not F1116C, I1117C, I1119C, or L1120C-are accessible to externally applied MTS reagents [10].
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ABCC7 p.Thr1121Cys 23955087:122:37
status: NEW123 Figure 3 confirms that application of external MTSES (200 bc;M) following channel activation with cAMP-stimulatory cocktail caused a decrease in whole cell current amplitude in T1115C, S1118C, and T1121C, but not in I1112C.
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ABCC7 p.Thr1121Cys 23955087:123:200
status: NEW