Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator)

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basi s of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the pres ent study, to determine the structural basis for the ATPase activity of CFT R, we assessed the effect of mutations within the '"Walker A" consensus mot ifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysi ne residue in the "Walker A" motif of either the first nucleotide binding f old (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibite d the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding fo lds of CFTR are functioning cooperatively in catalysis. However, the rate o f channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly cou pled to channel gating as previously hypothesized.