ABCMdb

ABCC7 p.Ala107Cys

CF databases:	c.320C>A , p.Ala107Asp (CFTR1) ? , c.320C>G , p.Ala107Gly (CFTR1) ? ,
Predicted by SNAP2:	C: N (61%), D: N (53%), E: N (57%), F: D (71%), G: N (82%), H: D (66%), I: N (61%), K: N (57%), L: N (61%), M: D (59%), N: N (72%), P: D (53%), Q: N (72%), R: N (61%), S: N (87%), T: N (82%), V: N (72%), W: D (71%), Y: D (71%),
Predicted by PROVEAN:	C: N, D: N, E: N, F: N, G: N, H: D, I: 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,

[switch to compact view]
Comments [show]

None has been submitted yet.

Publications
[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.

Comments [show]

None has been submitted yet.

Sentences [show]
No. Sentence Comment
8 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. Login to comment
105 Further addition of a specific CFTR inhibitor, CFTRinh-172 (39,40), caused a minor decrease of the residual current, indicating a drastic reduction of I106C-CFTR currents by external MTSES. Similar results were obtained for A107C- and Y109C-CFTR except that the magnitude of inhibition for A107C-CFTR is significantly smaller (Fig. 3 C). Login to comment
113 The similarity between TM1 and TM6 was reinforced by the following results with A107C mutant channels. Login to comment
114 When recording whole-cell A107C-CFTR current with a chloride gradient (24 mM internal and 156 mM external [Cl ]), we found that instead of an expected outward rectified I-V curve due to this imposed concentration gradient, the observed I-V relationship shows significant inward rectification (Fig. 4 A). Login to comment
124 We next adopted the similar strategy used in Liu et al. (42) to test if A107C-CFTR can be modulated with baths of different pH. Login to comment
125 In Fig. 4 B, A107C-CFTR channel currents were first activated with forskolin in the bath solution with a pH of 7.4; after the current level stabilized, the bath was switched to forskolin-containing solution with a pH of 6 or 8. Login to comment
143 FIGURE 4 Effects of external MTSES and pH on A107C-CFTR. Login to comment
144 (A) Whole-cell A107C-CFTR currents in response to external MTSES (left) and I-V curves extracted from the whole-cell recording as marked. Login to comment
145 (B) Effects of acidic or alkaline pH on the whole-cell A107C-CFTR currents (left). Login to comment