Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR), when phosphorylated, undergoes conformational change, and the chloride cha nnel opens. We investigated the contribution of R domain conformation, apar t:from the changes induced by phosphorylation, to channel opening, by testi ng 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 phosp horylation, 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 numb er of channel openings, not open time. Three highly conserved proline resid ues in the R domain, at positions 7401 750, and 759, were considered as can didate targets for cyclophilin A, Mutations of these prolines to alanines ( P3A mutant) resulted in a channel unresponsive to cyclophilin A but with po re 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 chan nel openings and not open time. Mutation of each of the proline residues se parately and in pairs demonstrated that all three proline mutations are req uired for maximal P-o. When P3A was expressed in 293 HEK cells and tested b y SPQ assay, chloride efflux was significantly increased compared to cells transfected with wild-type CFTR. Thus, treatments favoring the trans-peptid yl conformation about conserved proline residues in the R domain of CFTR af fect openings of CFTR, above and beyond the effect of PKA phosphorylation.