Characterization of K+ and Cl- conductances in apical membrane vesicles from stimulated rabbit oxyntic cells

Abstract

K+ and Cl- conductance pathways in apical membrane vesicles (SA vesicles) of stimulated oxyntic cells have been characterized. SA vesicles were prepared from rabbit fundic mucosa after stimulation of acid secretion with histamine. Conductive K+ and Cl- fluxes were assayed by several methods: by their effects on pH gradient formation by endogenous H(+)-K(+)-ATPase, by the protonophore-induced dissipation of preformed pH gradients, and by the effects of channel blockers. pH gradient formation by H(+)-K(+)-ATPase required K+ and was greatly reduced when the permeant anion chloride was replaced by gluconate or sulfate. In the presence of 75 mM K+, 1 mM Cl- was sufficient for generation of near maximal pH gradients, as was 5 mM K+ in the presence of 75 mM Cl-. At all K+ and Cl- concentrations tested, the ATP-generated formation of pH gradients was inhibited and the dissipation of these pH gradients stimulated by the protonophore tetrachlorosalicylanilide (TCS). Similar effects of TCS were also seen when Cl- was replaced by impermeant anions. Both processes were blocked by the K+ channel inhibitor Ba2+. The Ki for Ba2+ inhibition of pH gradient formation was 1.5 microM at 5 mM K+ and was proportional to the 3rd power of the K+ concentration. At 75 mM K+ the Cl- channel blocker diphenylamine-2-carboxylate inhibited ATP-dependent pH gradient formation when the Cl- concentration was 1 mM; however, when the Cl- concentration was greater than 5 mM no inhibition was observed. The membrane potential-sensitive dye DISC (5) was used to measure membrane potential generated by K+ gradients.(ABSTRACT TRUNCATED AT 250 WORDS)