title: Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings.
abstract: 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.
authors.affiliation: Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
authors.forename: J
authors.initials: J
authors.lastname: Xie
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: P B
authors.initials: PB
authors.lastname: Davis
authors.forename: J
authors.initials: J
authors.lastname: Ma
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D002460:Cell Line; D017124:Conserved Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D019696:Peptidylprolyl Isomerase / Q000494:pharmacology; D010766:Phosphorylation; D011336:Probability; D011392:Proline; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011392:Proline; D019696:Peptidylprolyl Isomerase; D000409:Alanine