title: 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(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.
authors.affiliation: Institute of Chemistry, Technical University of Berlin, Berlin, Germany.
authors.forename: Katharina L
authors.initials: KL
authors.lastname: Dürr
authors.forename: Neslihan N
authors.initials: NN
authors.lastname: Tavraz
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D000818:Animals; D001665:Binding Sites; D002412:Cations / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D005470:Fluorometry; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D009865:Oocytes / Q000201:enzymology*; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D012413:Rubidium / Q000378:metabolism; D013270:Stomach / Q000201:enzymology*; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D002412:Cations; D017506:H(+)-K(+)-Exchanging ATPase; D012413:Rubidium