cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium

J. Copello, T. A. Heming, Y. Segal, Luis Reuss

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30 Scopus citations

Abstract

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 > SO42− - gluconate - cyclamate. GaCl. l was not affected by the putative Cl channel blockers Cu 2+, 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 ~ 100 mM Cl in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of ~ 10 pS. The less frequent channel was outward rectifying with slope conductances of ~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 (~ 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 CIchannels 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.

Original languageEnglish (US)
Pages (from-to)177-199
Number of pages23
JournalJournal of General Physiology
Volume102
Issue number2
DOIs
StatePublished - Aug 1 1993

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