Interaction of nucleotides with membrane-associated cystic fibrosis transmembrane conductance regulator.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cytosolic nucleotides and by cAMP-dependent phosphorylation. In excised membrane patches, CFTR Cl- channel activity requires hydrolyzable nucleotides and Mg2+, and is inhibited by ADP. We examined the interactions between CFTR and nucleotides using 8-azidoadenosine 5'-triphosphate (8-N3-ATP), 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-32P]8-N3ATP specifically photolabeled CFTR, with half-maximal labeling at 10 microM 8-N3ATP in the presence of Mg2+ and 100 microM in the absence of Mg2+. The 8-N3ATP also substituted for ATP in activating CFTR Cl- channels, indicating that it interacts with the active site(s). Both ATP and GTP prevented photolabeling with half-maximal inhibition at 1 mM. ADP and adenyl-5'-yl imidodiphosphate (AMP-PNP) prevented photolabeling but at much higher concentrations, whereas AMP did not inhibit photolabeling at concentrations of up to 100 mM. Phosphorylation of CFTR was not a prerequisite for nucleotide binding. These results demonstrate that CFTR interacts directly with nucleotides at concentrations that regulate CFTR Cl- channel activity.
Travis
S M
SM
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa, College of Medicine, Iowa City 52242.
Carson
M R
MR
Ries
D R
DR
Welsh
M J
MJ
D000227:Adenine Nucleotides / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000345:Affinity Labels; D000818:Animals; D001386:Azides; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002478:Cells, Cultured; D018118:Chloride Channels; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009036:Moths; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism
D000227:Adenine Nucleotides; D000345:Affinity Labels; D001386:Azides; C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008274:Magnesium