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 (G^a_Cl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (G^a) was 0.17 mS·cm^-2, primarily ascribable to G^a_K. With elevation of cell cAMP to maximum levels, G^a increased to 6.7 mS·cm^-2 and became anion selective, with the permeability sequence SCN^- > NO^-₃ > I^- > Br^- > Cl^- ≫ SO^2-₄ ∼ gluconate ∼ cyclamate. G^a_Cl 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 ∼ 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 (P_o) 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 G^a_Cl. 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 P_o to 0.5, close to expectations from G^a_Cl. 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.