Phosphate stimulates CFTR Cl- channels.
Cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels appear to be regulated by hydrolysis of ATP and are inhibited by a product of hydrolysis, ADP. We assessed the effect of the other product of hydrolysis, inorganic phosphate (P(i)), on CFTR Cl- channel activity using the excised inside-out configuration of the patch-clamp technique. Millimolar concentrations of P(i) caused a dose-dependent stimulation of CFTR Cl- channel activity. Single-channel analysis demonstrated that the increase in macroscopic current was due to an increase in single-channel open-state probability (po) and not single-channel conductance. Kinetic modeling of the effect of P(i) using a linear three-state model indicated that the effect on po was predominantly the result of an increase in the rate at which the channel passed from the long closed state to the bursting state. P(i) also potentiated activity of channels studied in the presence of 10 mM ATP and stimulated Cl- currents in CFTR mutants lacking much of the R domain. Binding studies with a photoactivatable ATP analog indicated that Pi decreased the amount of bound nucleotide. These results suggest that P(i) increased CFTR Cl- channel activity by stimulating a rate-limiting step in channel opening that may occur by an interaction of P(i) at one or both nucleotide-binding domains.
Carson
M R
MR
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Travis
S M
SM
Winter
M C
MC
Sheppard
D N
DN
Welsh
M J
MJ
D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D018118:Chloride Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D009154:Mutation; D010710:Phosphates / Q000494:pharmacology*; D013431:Sulfates / Q000494:pharmacology; D014162:Transfection
D001386:Azides; C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D010710:Phosphates; D013431:Sulfates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate