Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites

The CFTR chloride channel is regulated by phosphorylation by protein kinase s, especially PKA, and by nucleotides interacting with the two nucleotide b inding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches fi-om Xenopus oocytes expressing human epithelial cystic fibrosis transmemb rane conductance regulator (CFTR) were tested for their chloride conductanc e in response to the application of PKA and nucleotides. Rapid changes in t he concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosph ate (AMP-PNP), its photolabile derivative ATP-P-3-[1-(2-nitrophennyl)ethyl] ester, or ADP led to changes in chloride conductance with characteristic ti me constants, which reflected interaction of CFTR with these nucleotides, T he conductance changes of strongly phosphorylated channels were slower than there of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay whereas ATP-P-3-[1-(2-nitrophenyl)ethyl]est er only decelerated the conductance increase upon ATP addition. ADP deceler ated the conductance increase upon;ATP addition and accelerated the conduct ance decay upon ATP withdrawal. The results present the first direct eviden ce that AMP-PNP binds to two sites on the CFTR. The effects of ADP also sug gest two different binding sites because of the two different modes of inhi bition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either re flect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) o r allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NB D-A.