THE 2 NUCLEOTIDE-BINDING DOMAINS OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) HAVE DISTINCT FUNCTIONS IN CONTROLLING CHANNEL ACTIVITY

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- cha nnel contains two cytoplasmic nucleotide-binding domains (NBDs), After phosphorylation of the R domain, ATP interacts with the NBDs to regul ate channel activity, To learn how the NBDs regulate channel function, we used the patch-clamp technique to study CFTR and variants which co ntained site-directed mutations in the conserved Walker A motif lysine residues in either NBD1 (K464A), NBD2 (K1250A and K1250M), or both NB Ds simultaneously (K464A/K1250A), Studies in related proteins suggest that such mutations slow the rate of ATP hydrolysis, These mutations d id not alter the conductive properties of the channel or the requireme nt for phosphorylation and ATP to open the channel, However, all mutat ions decreased open state probability, Mutations in NBD1 decreased the frequency of bursts of activity, whereas mutations in NBD2 and mutati ons in both NBDs simultaneously prolonged bursts of activity, as well as decreased the frequency of bursts, These results could not be attri buted to altered binding of nucleotide because none of the mutants stu died had reduced 8-N(3)ATP binding. These data suggest that the two NB Ds have distinct functions in channel gating; ATP hydrolysis at NBD1 i nitiates a burst of activity, and hydrolysis at NBD2 terminates a burs t.