cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.
Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.
Copello
J
J
Department of Physiology, University of Texas Medical Branch, Galveston 77555.
Heming
T A
TA
Segal
Y
Y
Reuss
L
L
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D001965:Bromides / Q000493:pharmacokinetics; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002463:Cell Membrane Permeability / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D000242:Cyclic AMP / Q000032:analysis / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology / Q000648:ultrastructure; D005704:Gallbladder / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D007455:Iodine / Q000493:pharmacokinetics; D008839:Microelectrodes; D009338:Necturus maculosus / Q000502:physiology*; D013806:Theophylline / Q000494:pharmacology
D001965:Bromides; D018118:Chloride Channels; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D003300:Copper; D000255:Adenosine Triphosphate; D007455:Iodine; D013806:Theophylline; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid