Control of gastric H,K-atpase activity by cations, voltage and intracellular pH analyzed by voltage clamp fluorometry in xenopus oocytes

Abstract

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 1P and E 2P states and measured Rb + uptake under various ionic and pH conditions. The steady-state E 1P/E 2P 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 1P/E 2P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V 0.5, the voltage, at which the E 1P/E 2P ratio is 50:50, by -100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E 1P→E 2P 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 1P→E 2P 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 1P/E 2P distribution indicating that Na + ions can act as surrogates for protons regarding the E 2P→E 1P 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. © 2012 Dürr et al.