ABCMdb

ABCC7 p.Thr757Ser

Predicted by SNAP2:	A: N (82%), C: N (66%), D: N (78%), E: N (87%), F: D (53%), G: N (66%), H: N (78%), I: N (57%), K: N (78%), L: N (57%), M: N (61%), N: N (87%), P: N (82%), Q: N (82%), R: N (66%), S: N (93%), V: N (66%), W: D (63%), Y: N (57%),
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, S: N, V: N, W: D, Y: N,

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Publications
[hide] Conformation, independent of charge, in the R doma... Biophys J. 2000 Mar;78(3):1293-305. Xie J, Zhao J, Davis PB, Ma J
Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings.
Biophys J. 2000 Mar;78(3):1293-305., [PMID:10692317]

Abstract [show]
The R domain of cystic fibrosis transmembrane conductance regulator (CFTR), when phosphorylated, undergoes conformational change, and the chloride channel opens. We investigated the contribution of R domain conformation, apart from the changes induced by phosphorylation, to channel opening, by testing the effect of the peptidyl-prolyl isomerase, cyclophilin A, on the CFTR channel. When it was applied after the channel had been opened by PKA phosphorylation, cyclophilin A increased the open probability of wild-type CFTR (from P(o) = 0.197 +/- 0.010 to P(o) = 0.436 +/- 0. 029) by increasing the number of channel openings, not open time. Three highly conserved proline residues in the R domain, at positions 740, 750, and 759, were considered as candidate targets for cyclophilin A. Mutations of these prolines to alanines (P3A mutant) resulted in a channel unresponsive to cyclophilin A but with pore properties similar to the wild type, under strict control of PKA and ATP, but with significantly increased open probability (P(o) = 0.577 +/- 0.090) compared to wild-type CFTR, again due to an increase in the number of channel openings and not open time. Mutation of each of the proline residues separately and in pairs demonstrated that all three proline mutations are required for maximal P(o). When P3A was expressed in 293 HEK cells and tested by SPQ assay, chloride efflux was significantly increased compared to cells transfected with wild-type CFTR. Thus, treatments favoring the trans-peptidyl conformation about conserved proline residues in the R domain of CFTR affect openings of CFTR, above and beyond the effect of PKA phosphorylation.

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No. Sentence Comment
33 Site-specific mutations were constructed following the manufacturer`s instructions using the following three mutagenesis oligonucleotides (showed 5Ј to 3Ј, with mutated base underlined): P740A, G CTC AGA ATC TGC TAC TAA GGA CAG C (RsaI enzyme restriction site is destroyed); P750A, G GCT GAT GCG AGC CAG TAT CGC CTC (BsrI site is created); P759A/T757S, C CTG AAG CGT GGC CGG AGA GCT GAT (BsaHI site is created and BsrI site is destroyed). Login to comment
35 Although mutation was intended to affect only proline residues, upon sequencing, an additional conservative mutation, T757S, was noted. Login to comment
36 Subsequently, the individual prolines were mutated in sequence, with and without the T757S mutation, and their function was examined. Login to comment
37 No difference in channel activity was produced for any mutant by the T757S substitution. Login to comment
38 Data presented in this paper for mutants containing P759A mutation are for constructs that also contain the unexpected T757S mutation. Login to comment
34 Site-specific mutations were constructed following the manufacturer`s instructions using the following three mutagenesis oligonucleotides (showed 5b18; to 3b18;, with mutated base underlined): P740A, G CTC AGA ATC TGC TAC TAA GGA CAG C (RsaI enzyme restriction site is destroyed); P750A, G GCT GAT GCG AGC CAG TAT CGC CTC (BsrI site is created); P759A/T757S, C CTG AAG CGT GGC CGG AGA GCT GAT (BsaHI site is created and BsrI site is destroyed). Login to comment
39 Data presented in this paper for mutants containing P759A mutation are for constructs that also contain the unexpected T757S mutation. Login to comment