IDENTIFICATION AND PARTIAL CHARACTERIZATION OF A DOMAIN IN CFTR THAT MAY BIND CYCLIC-NUCLEOTIDES DIRECTLY

Background: The cystic fibrosis transmembrane conductance regulator (C FTR) is a-chloride channel that is activated by cAMP-dependent phospho rylation. CFTR channel activity is also stimulated by cGMP-dependent p rotein kinase and protein kinase C. Results: Here,we show that CFTR ch annel activation by cGMP may also occur directly. In oocytes from one third of Xenopus donors, the activation of CFTR by cGMP averaged 87 % of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current-voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP a nd cGMP was not additive, suggesting that both cyclic nucleotides acti vate the same channel; cGMP was one order of magnitude more potent tha n cAMP, and its action was insensitive to protein kinase inhibitors. A nalysis of the amino-acid sequence of CFTR revealed a domain in the am ino-terminal portion of the third cytoplasmic loop that resembles a cl ass of cyclic-nucleotide-binding domains related to that of the catabo lite-gene activator protein, CAP. Two CFTR residues in this domain - V al397 and Lys420 - were identified which, when changed to alanine, alt ered the response to cGMP independently of the response to cAMP. Concl usions: We conclude that direct cyclic nucleotide binding may play a r ole in channel gating of CFTR. The cGMP-binding domain may provide a u seful target for pharmacologic intervention in cystic fibrosis.