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Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in  Xenopus  oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E 1 P and E 2 P states and measured Rb +  uptake under various ionic and pH conditions. The steady-state E 1 P/E 2 P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb +  uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E 1 P/E 2 P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V 0.5 , the voltage, at which the E 1 P/E 2 P ratio is 50∶50, by −100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E 1 P→E 2 P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb +  uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E 1 P→E 2 P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na +  profoundly alters the voltage-dependent E 1 P/E 2 P distribution indicating that Na +  ions can act as surrogates for protons regarding the E 2 P→E 1 P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively.

Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes