ABCC7 p.Gly1127Cys
| CF databases: |
c.3380G>A
,
p.Gly1127Glu
(CFTR1)
?
, This mutation has been found in one among 50 non-[delta]F508 CF chromosomes.
|
| Predicted by SNAP2: | A: D (71%), C: D (80%), D: D (91%), E: D (85%), F: D (91%), H: D (91%), I: D (80%), K: D (95%), L: D (91%), M: D (85%), N: D (75%), P: D (95%), Q: D (91%), R: D (91%), S: D (80%), T: D (85%), V: D (85%), W: D (95%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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Comments [show]
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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.
Comments [show]
None has been submitted yet.
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.
X
ABCC7 p.Gly1127Cys 19381710:71:236
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.
X
ABCC7 p.Gly1127Cys 19381710:72:166
status: NEW84 Unitary currents at depolarized voltages were significantly decreased in S1118C, T1121C, T1122C and G1127C and significantly increased in V1129C (Fig. 5b).
X
ABCC7 p.Gly1127Cys 19381710:84:100
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).
X
ABCC7 p.Gly1127Cys 19381710:85:134
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).
X
ABCC7 p.Gly1127Cys 19381710:91:88
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).
X
ABCC7 p.Gly1127Cys 19381710:118:40
status: NEW130 Block was not significantly altered in either T1122C or G1127C (data not shown).
X
ABCC7 p.Gly1127Cys 19381710:130:56
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).
X
ABCC7 p.Gly1127Cys 19381710:133:116
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).
X
ABCC7 p.Gly1127Cys 19381710:158:100
status: NEW161 a S1118C (d), T1121C (j), T1122C (), G1127C (h); b V1129C (m), I1131C (r), I1132C (.).
X
ABCC7 p.Gly1127Cys 19381710:161:38
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.
X
ABCC7 p.Gly1127Cys 19381710:183:73
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).
X
ABCC7 p.Gly1127Cys 19381710:188:87
status: NEW200 Mutations S1118C, T1122C and G1127C also altered the anion selectivity of CFTR, significantly increasing SCN- per- meability (Fig. 11), which is consistent with changes in pore structure and function.
X
ABCC7 p.Gly1127Cys 19381710:200:29
status: NEW