Regulation of CFTR Cl- conductance in secretion by cellular energy levels.
Recent studies suggested dual regulation of the Cl- conductance (GCl) affected in cystic fibrosis, one by protein kinase A-dependent phosphorylation and a second by low-affinity ATP binding. We proposed that ATP binding may couple the transport demands to the energy level of the cell. In the present study we examined this hypothesis further in a purely secretory function using the epithelial cell line T84. We used a depletion-permeabilization protocol on cells grown on permeable supports to deplete the cells of endogenous ATP and to provide access to the intracellular compartment for the impermeable nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and ATP. In contrast to non-depleted permeabilized cells, which responded to 0.1 mM cAMP with an increase in transepithelial potential (delta Vt = 29.8 +/- 3.0 mV, n = 4) and conductance (delta Gt = 1.23 +/- 0.54 mS/cm2, n = 4), addition of cAMP to ATP-depleted cells resulted in insignificant changes in Vt (delta Vt = 0.7 +/- 0.2 mV, n = 26; P < 0.05) and Gt (delta Gt = 0.020 +/- 0.003 mS/cm2, n = 26; P < 0.05). However, the cAMP response was restored by addition of 5 mM ATP (delta Vt = 21.7 +/- 1.5 mV, n = 26; delta Gt = 0.59 +/- 0.06 mS/cm2, n = 26). ATP dose-response experiments, taken together with the effect of cAMP with and without ATP, suggest that phosphorylation is necessary, but not sufficient, for activation. The data provide evidence for a second level of regulation of GCl, which requires high concentrations of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Bell
C L
CL
Division of Biomedical Sciences, University of California, Riverside 92521.
Quinton
P M
PM
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003847:Deoxyglucose / Q000494:pharmacology; D004553:Electric Conductivity / Q000187:drug effects; D004734:Energy Metabolism; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors
C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D000242:Cyclic AMP; D000241:Adenosine