ABCC7 p.Ser1149Cys
| Predicted by SNAP2: | A: N (57%), C: D (59%), D: D (71%), E: D (75%), F: D (85%), G: N (61%), H: D (85%), I: D (66%), K: D (75%), L: D (66%), M: D (71%), N: N (57%), P: D (80%), Q: N (53%), R: D (71%), T: N (78%), V: D (63%), W: D (91%), Y: D (85%), |
| 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, T: N, V: N, W: N, Y: N, |
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[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
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).
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ABCC7 p.Ser1149Cys 21796338:80:305
status: NEW86 However, unlike previous findings in TM6 [9], we observed a third pattern for cysteines introduced closest to the putative cytoplasmic end of TM12 (V1147C, N1148C, and S1149C).
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ABCC7 p.Ser1149Cys 21796338:86:168
status: NEW87 Here, macroscopic current amplitude was decreased by MTSES application but either decreased (V1147C, S1149C) or not significantly affected (N1148C) by MTSET application.
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ABCC7 p.Ser1149Cys 21796338:87:101
status: NEW[hide] Structural basis for the channel function of a deg... J Gen Physiol. 2011 Nov;138(5):495-507. Bai Y, Li M, Hwang TC
Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).
J Gen Physiol. 2011 Nov;138(5):495-507., [PMID:22042986]
Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel.
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No. Sentence Comment
209 However, although they detected reactivity in N1138C and S1149C, we found that these sites appear nonreactive.
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ABCC7 p.Ser1149Cys 22042986:209:57
status: NEW