Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis

Opening and closing of the cystic fibrosis transmembrane conductance regula tor (CFTR) Cl- channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by th e NBDs is required for normal gating. the influence of ATP binding versus h ydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that eve n in the absence of Mg2+. ATP could support channel activity, albeit at a r educed level compared with the presence of Mg2+. Application of ATP with a divalent cation. including the poorly hydrolyzed CaATP complex, increased t he rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-depe ndent opening. These data suggest that ATP alone can open the channel and t hat divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested t hat divalent cations;also stabilize the open state. In wild-type CFTR, CaAT P generated a long duration open state, whereas ATP alone did not. With a C FTR variant in which hydrolysis was disrupted. MgATP. but not ATP alone, pr oduced long openings. These results suggest a gating cycle for CFTR in whic h ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hyd rolysis by either NBD can gate the channel.