ABCMdb

ABCC7 p.Ile1131Cys

CF databases:	c.3391A>G , p.Ile1131Val (CFTR1) ? , This variant was found by DGGE analysis and characterized by DNA sequencing. It was found in a sample of 100 CFTR genes from 50 apparently healthy random individuals from Central Italy.
Predicted by SNAP2:	A: N (57%), C: N (66%), D: D (75%), E: D (71%), F: N (57%), G: D (71%), H: D (66%), K: D (71%), L: N (93%), M: N (66%), N: D (66%), P: D (75%), Q: N (57%), R: D (71%), S: N (53%), T: N (61%), V: N (66%), W: D (71%), Y: D (59%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: N, G: D, H: D, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: D,

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Publications
[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. Login to comment
75 As described above (Fig. 2), six mutants had I-V relationships that were sensitive to both MTSET and MTSES and only one (I1131C) was altered by MTSET but not MTSES. Login to comment
82 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). Login to comment
86 No changes in i-V shape were observed for I1131C or I1132C. Login to comment
91 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). Login to comment
114 Note that the rectification ratio in six cysteine mutants is significantly affected by both MTSET and MTSES (indicated by asterisks), whereas in a seventh (I1131C) it is significantly altered by MTSET but not by MTSES voltages as an indicator, Fig. 8 shows that MTS reagents had charge-dependent effects on i-V relationship shape that broadly mirrored those observed in the macroscopic I-V relationships (see Fig. 3c). Login to comment
119 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). Login to comment
124 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. Login to comment
133 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). Login to comment
161 a S1118C (d), T1121C (j), T1122C (), G1127C (h); b V1129C (m), I1131C (r), I1132C (.). Login to comment
187 * Significant difference from wild-type (P \ 0.05) I1131C, I1132C) represents mutations at the outermost mouth of the pore. Login to comment
191 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. 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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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). Login to comment
126 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. Login to comment

[hide] Cystic fibrosis transmembrane conductance regulato... Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7. Norimatsu Y, Ivetac A, Alexander C, Kirkham J, O'Donnell N, Dawson DC, Sansom MS
Cystic fibrosis transmembrane conductance regulator: a molecular model defines the architecture of the anion conduction path and locates a "bottleneck" in the pore.
Biochemistry. 2012 Mar 20;51(11):2199-212. Epub 2012 Mar 7., [PMID:22352759]

Abstract [show]
We developed molecular models for the cystic fibrosis transmembrane conductance regulator chloride channel based on the prokaryotic ABC transporter, Sav1866. Here we analyze predicted pore geometry and side-chain orientations for TM3, TM6, TM9, and TM12, with particular attention being paid to the location of the rate-limiting barrier for anion conduction. Side-chain orientations assayed by cysteine scanning were found to be from 77 to 90% in accord with model predictions. The predicted geometry of the anion conduction path was defined by a space-filling model of the pore and confirmed by visualizing the distribution of water molecules from a molecular dynamics simulation. The pore shape is that of an asymmetric hourglass, comprising a shallow outward-facing vestibule that tapers rapidly toward a narrow "bottleneck" linking the outer vestibule to a large inner cavity extending toward the cytoplasmic extent of the lipid bilayer. The junction between the outer vestibule and the bottleneck features an outward-facing rim marked by T338 in TM6 and I1131 in TM12, consistent with the observation that cysteines at both of these locations reacted with both channel-permeant and channel-impermeant, thiol-directed reagents. Conversely, cysteines substituted for S341 in TM6 or T1134 in TM12, predicted by the model to lie below the rim of the bottleneck, were found to react exclusively with channel-permeant reagents applied from the extracellular side. The predicted dimensions of the bottleneck are consistent with the demonstrated permeation of Cl(-), pseudohalide anions, water, and urea.

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Sentences [show]
No. Sentence Comment
177 It is noteworthy that in several cases (e.g., I1131C) application of MTSES- , which carries a net negative charge, led to an increase in macroscopic conductance. Login to comment

[hide] Locating a Plausible Binding Site for an Open Chan... Mol Pharmacol. 2012 Aug 24. Norimatsu Y, Ivetac A, Alexander C, O'Donnell N, Frye L, Sansom MS, Dawson DC
Locating a Plausible Binding Site for an Open Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator.
Mol Pharmacol. 2012 Aug 24., [PMID:22923500]

Abstract [show]
High-throughput screening has led to the identification of small-molecule blockers of the CFTR chloride channel, but the structural basis of blocker binding remains to be defined. We recently developed molecular models of the CFTR channel based on homology to the bacterial transporter, Sav1866, that could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker like GlyH-101, thought to act by entering the channel from the extracellular side. As a more stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual ligand docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest scoring, docked position was near two pore-lining residues, F337 and T338, and the rate of reaction of anionic thiol-directed reagents with cysteines substituted at either of these positions was slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (F342) was replaced with alanine, the apparent affinity of the blocker increased by approximately 200 fold. A Molecular Mechanics-Generalized Born/Surface Area (MM-GB/SA) analysis of GlyH-101 binding predicted that substitution of F342 with alanine would substantially increase blocker affinity, primarily due to decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

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Sentences [show]
No. Sentence Comment
151 We also studied the reaction of I1131C CFTR with MTSES- . Login to comment
154 In fact, no occlusion of I1131C was observed with 10 µM GlyH-101, despite more than 80% block of the I1131C CFTR current. Login to comment
155 The rate of reaction between MTSES- and I1131C CFTR was actually somewhat greater in the presence of GlyH-101 (Figure 7A). Login to comment
156 The reaction rates of charged reagents with I1131C CFTR would be influenced by relative positions of nearby charges, which may change slightly in the event of binding of GlyH-101. Login to comment
159 The presence of negative charges near I1131 is consistent with the observed slow reaction of I1131C CFTR with MTSES- (20 M-1 sec-1 ; Figure 7) which is more than 100-fold less than that seen for a Cys at 338 (3.3 x 103 M-1 sec-1 ; Figure 6). Login to comment
160 As reported previously (Norimatsu et al., 2012) the macroscopic conductance of I1131C CFTR was increased by depositing the negatively charged sulfonic acid group via reaction with MTSES- . Login to comment
161 This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels similar to that observed for R352C CFTR by Bai et al., (2010). Login to comment
234 We also studied the reaction of the I1131C CFTR with MTSESafa; . Login to comment
237 No occlusion of I1131C was observed with 10 òe;M GlyH-101, despite more than 80% blockade of the I1131C CFTR current. Login to comment
238 The rate of reaction between MTSESafa; and the I1131C CFTR was actually somewhat greater in the presence of GlyH-101 (Fig. 7A). Login to comment
239 The rates of reactions of charged reagents with the I1131C CFTR would be influenced by the relative positions of nearby charges, which might change slightly upon GlyH-101 binding. Login to comment
242 The presence of negative charges near Ile1131 is consistent with the observed slow reaction of the I1131C CFTR with MTSESafa; (20 Mafa;1 safa;1 ) (Fig. 7), which is more than 100-fold less than the rate seen with a cysteine at position 338 (3.3 afb; 103 Mafa;1 safa;1 ) (Fig. 6). Login to comment
243 As reported previously (Norimatsu et al., 2012), the macroscopic conductance of the I1131C CFTR was increased by deposition of the negatively charged sulfonic acid group through reaction with MTSESafa; . Login to comment
244 This result is most likely a reflection of an increase in the open probability of I1131C CFTR channels, similar to that observed for the R352C CFTR (Bai et al., 2010). Login to comment
273 A, time courses of the reactions of the I1131C CFTR with 1 mM MTSESafa; in the presence and absence of 10 òe;M GlyH-101. Data points represent mean afe; S.E.M. (n afd; 3). Login to comment
274 Covalent labeling of the I1131C CFTR with MTSESafa; resulted in increases in conductance. Login to comment
278 The EC50 at 0 mV for GlyH-101 blockade for the I1131C CFTR was 0.86 afe; 0.016 òe;M (n afd; 3). Login to comment