Bursting activity of the cystic fibrosis transmembrane conductance regulator reveals intrinsic gating scheme

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel defective in the genetic disease cystic fibrosis (CF). Channel openings and closings, termed channel gating of CFTR is characterized by bursts of openings interrupted by brief shuts and separated by long closures between bursts. It is unclear how these short-lived shuts and sustained openings occur during CFTR bursting state and whether CF-associated mutations disrupt their properties. We address these questions by studying single-channel properties of CFTR at pHi 7.3 and 6.3. Compared to neutral, acidic pHi altered CFTR gating at bursting state with decreased open time constant to and increased fast closed time constant tcf. These alterations were not sensitive to ATP concentrations but nicely simulated by the C1 ↔ O ↔ C2 kinetic scheme. Moreover, studying CFTR variants demonstrates that the acid-sensitive alteration in to was abolished by site-directed mutations at two ATP binding sites (site 1 and site 2) in CFTR. In contrast, tcf regulation by acidic pHi was altered by mutants at site 1 but not site 2. CF mutant G1349D-CFTR at site 1 markedly reduced the value of tcf and to, whereas CF mutants G551D-CFTR at site 2 and DF508-CFTR showed small or no effects on them. Our data suggest that site 1 predominantly controls gating events during bursting state. This study provides a fundamental basis of CFTR gating mechanism suggesting how CF-associated mutations affect CFTR gating.