INTERACTION OF NUCLEOTIDES WITH MEMBRANE-ASSOCIATED CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- ch annel that is regulated by cytosolic nucleotides and by cAMP-dependent phosphorylation. In excised membrane patches, CFTR Cl- channel activi ty requires hydrolyzable nucleotides and Mg2+, and is inhibited by ADP . We examined the interactions between CFTR and nucleotides using 8-az idoadenosine 5'-triphosphate (8-N3ATP), a photoactivatable ATP analog. Because CFTR functions as a membrane ion channel, we studied CFTR in membranes of Sf9 insect cells. We found that [alpha-P-32]8-N3ATP speci fically photolabeled CFTR, with half-maximal labeling at 10 muM 8-N3AT P in the presence of Mg2+ and 100 muM in the absence of Mg2+. The 8-N3 ATP also substituted for ATP in activating CFTR Cl- channels, indicati ng that it interacts with the active site(s). Both ATP and GTP prevent ed photolabeling with half-maximal inhibition at 1 mM. ADP and adenyl- 5'-yl imidodiphosphate (AMP-PNP) prevented photolabeling but at much h igher concentrations, whereas AMP did not inhibit photolabeling at con centrations of up to 100 mM. Phosphorylation of CFTR was not a prerequ isite for nucleotide binding. These results demonstrate that CFTR inte racts directly with nucleotides at concentrations that regulate CFTR C l- channel activity.