ABCC7 p.Gly103Cys
| Predicted by SNAP2: | A: D (71%), C: D (71%), D: D (85%), E: D (91%), F: D (85%), H: D (85%), I: D (85%), K: D (91%), L: D (85%), M: D (85%), N: D (71%), P: D (85%), Q: D (80%), R: D (85%), S: N (61%), T: D (75%), V: D (85%), W: D (91%), Y: D (85%), |
| Predicted by PROVEAN: | A: N, C: D, D: D, E: D, F: D, H: D, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: D, V: D, W: D, Y: D, |
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[hide] Cysteine scanning of CFTR's first transmembrane se... Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048. Gao X, Bai Y, Hwang TC
Cysteine scanning of CFTR's first transmembrane segment reveals its plausible roles in gating and permeation.
Biophys J. 2013 Feb 19;104(4):786-97. doi: 10.1016/j.bpj.2012.12.048., [PMID:23442957]
Abstract [show]
Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.
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No. Sentence Comment
95 Although we failed to detect any currents from G103C-CFTR, all other seven cysteine-substituted constructs did not respond to cytoplasmic application of MTSES to a significant extent (Fig. 2).
X
ABCC7 p.Gly103Cys 23442957:95:47
status: NEW139 (C) Summary of whole-cell SCAM results on the eight residues, G103C-D110C.
X
ABCC7 p.Gly103Cys 23442957:139:62
status: NEW142 * indicates no detectable current for G103C-CFTR.
X
ABCC7 p.Gly103Cys 23442957:142:38
status: NEW265 Single star specifies no channel activity was detected for G103C.
X
ABCC7 p.Gly103Cys 23442957:265:59
status: NEW