ABCC7 p.Ile1132Cys
| Predicted by SNAP2: | A: N (72%), C: N (66%), D: D (80%), E: D (75%), F: D (66%), G: D (75%), H: D (66%), K: D (80%), L: N (72%), M: N (78%), N: D (71%), P: D (85%), Q: D (59%), R: D (80%), S: N (61%), T: N (66%), V: N (87%), W: D (80%), Y: D (66%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: 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.Ile1132Cys 19381710:71:263
status: NEW82 In contrast, I1131C and I1132C did not significantly affect the form of either the i-V relationship (Fig. 4b) or the I-V relationship (Fig. 3a).
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ABCC7 p.Ile1132Cys 19381710:82:24
status: NEW86 No changes in i-V shape were observed for I1131C or I1132C.
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ABCC7 p.Ile1132Cys 19381710:86:52
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.Ile1132Cys 19381710:91:115
status: NEW119 In contrast, the other three mutations (V1129C, I1131C, I1132C) led to no change or even a slight increase in unitary current amplitude (Fig. 5b) and more minor effects of MTS modification, resulting in no change or a small decrease in amplitude with MTSES and increases in amplitude to levels above wild-type with MTSET (Fig. 9b).
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ABCC7 p.Ile1132Cys 19381710:119:56
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.Ile1132Cys 19381710:124:69
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.Ile1132Cys 19381710:133:167
status: NEW161 a S1118C (d), T1121C (j), T1122C (), G1127C (h); b V1129C (m), I1131C (r), I1132C (.).
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ABCC7 p.Ile1132Cys 19381710:161:76
status: NEW187 * Significant difference from wild-type (P \ 0.05) I1131C, I1132C) represents mutations at the outermost mouth of the pore.
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ABCC7 p.Ile1132Cys 19381710:187:60
status: NEW191 Chloride conductance was further reduced by MTSES modification at these sites, indicating the detrimental effect of depositing a negative charge within the permeation pathway. Cysteine substitution at the outer mouth of the pore (V1129C, I1131C, I1132C) (Fig. 9b) had somewhat different effects.
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ABCC7 p.Ile1132Cys 19381710:191:246
status: NEW[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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No. Sentence Comment
90 A similar lack of effect following prolonged (>5 min) exposure to such high concentrations of both MTSES and MTSET was also observed in ten out of 19 cysteine-substituted mutants tested (I1131C, I1132C, L1133C, T1134C, L1135C, A1136C, M1137C, I1139C, L1143C, and A1146C).
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ABCC7 p.Ile1132Cys 21796338:90:195
status: NEW126 Previous work from our group suggested that externally applied MTS reagents could modify cysteines only in the outermost part of TM12, namely, I1131C and I1132C [11]; these same cysteines were insensitive to internally applied MTS reagents (Fig. 2), again consistent with impermeability to these reagents.
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ABCC7 p.Ile1132Cys 21796338:126:154
status: NEW