CFTR Cytosolic Loop Mutations Allosterically Promote ATP Independent Channel Gating

Abstract

CFTR channel gating normally depends on ATP binding and NBD dimerization. Optimal CFTR channel activation further requires phosphorylation of the R domain. How ATP binding at the NBDs and phosphorylation of the R domain regulate CFTR channel gating are not fully understood. In the present study, we demonstrate that mutations in the CFTR Cytosolic Loops (CL) markedly promote channel opening in the absence of ATP and NBD2, presumably by an allosteric mechanism. In excised inside-out patches, we observed that single or double mutations of K978 and K190 in CL 3 and 1 induced large ATP independent currents (5-70% of current before removing ATP). These mutant channels deactivated with a slow time constant (49.11±4.58 sec) when ATP was removed by Hexokinase/glucose and subsequent bath perfusion. A K978 point mutation greatly increased the ATP sensitivity of channel activation by decreasing the EC50 (by 8-fold) for ATP activation, which is consistent with the slow deactivation following ATP removal. K978 mutations markedly enhanced G551D channel activity, a disease mutant that fails to respond to ATP, and Δ1198-CFTR, a mutant that lacks NBD2, indicating that the K978 mutations affect channel gating downstream of NBD dimerization. Interestedly, R domain phosphorylation further stimulated K978/G551D and K978/Δ1198 combined mutants, indicating that the R domain regulates channel activity independently of NBD dimerization. Similarly, K978 mutations also increased the activation rate at low dose (3 U/ml) of PKA, indicating that K978 mutations also enhance the PKA sensitivity of channel activation. Our results support an allosteric gating mechanism in which loops 1 and 3 functionally link ATP binding and NBD dimerization to CFTR channel opening.