cAMP-activated Cl channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells.
Retrovirus-mediated transfection of cDNA for the cystic fibrosis (CF) gene into the CF pancreatic cell line, CFPAC-1, confers adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of Cl conductance. We used patch-clamp techniques to identify the single-channel basis of this conductance pathway and to study its properties. Forskolin or cAMP activated Cl channels with a conductance of 9 +/- 1 pS in 26 of 62 cell-attached patches of cystic fibrosis transmembrane conductance regulator (CFTR)-transfected CFPAC-1 cells. The current-voltage (I-V) relation showed slight outward rectification (chord conductance of 10 +/- 2 pS at +80 mV vs. 7 +/- 1 pS at -80mV) with high Cl concentrations (170 mM) in the pipette solution. Channel kinetics were voltage sensitive, with longer openings at positive clamp voltages. Channel properties were unaffected by the substitution of N-methyl-D-glucamine for pipette Na or by the addition of disulfonic stilbenes (100 microM DNDS or DIDS) to the pipette. The channels usually inactivated within seconds of patch excision, but in three of nine patches, activity could be maintained by addition of the catalytic subunit of protein kinase A and ATP. With equal Cl concentrations on both membrane surfaces, the single-channel I-V relation was linear, suggesting that the outward rectification of the cell-attached channel is due to a pipette-to-cell Cl gradient. Anion substitution on the extracellular side of the membrane indicates a halide permselectivity of Br approximately Cl greater than I.(ABSTRACT TRUNCATED AT 250 WORDS)
Cliff
W H
WH
Department of Physiology, University of Alabama, Birmingham 35294.
Schoumacher
R A
RA
Frizzell
R A
RA
D002461:Cell Line, Transformed; D018118:Chloride Channels; D000242:Cyclic AMP / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000378:metabolism* / Q000473:pathology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004553:Electric Conductivity; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D006219:Halogens / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D010179:Pancreas / Q000378:metabolism* / Q000473:pathology; D014162:Transfection
D018118:Chloride Channels; D006219:Halogens; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP