AJP - Cell Physiology, Vol 262, Issue 5 C1154-C1160, Copyright © 1992 by American Physiological Society
cAMP-activated Cl channels in CFTR-transfected cystic fibrosis pancreatic epithelial cells
W. H. Cliff, R. A. Schoumacher and R. A. Frizzell
Department of Physiology, University of Alabama, Birmingham 35294.
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)
