control of gastric h,k-atpase activity by cations, voltage and intracellular ph analyzed by voltage clamp fluorometry in xenopus oocytes控制胃h,k-atpase活动通过阳离子,电压和细胞内的ph值分析了电压钳位在非洲爪蟾蜍卵母细胞荧光测定术.pdf

Control of Gastric H,K-ATPase Activity by Cations, Voltage and Intracellular pH Analyzed by Voltage Clamp Fluorometry in Xenopus Oocytes ¨ ¤ Katharina L. Durr , Neslihan N. Tavraz, Thomas Friedrich* Institute of Chemistry, Technical University of Berlin, Berlin, Germany 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 P and E P states and measured Rb+ uptake under various ionic and pH 1 2 conditions. The steady-state E P/E P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the 1 2 Rb+ uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E P/E P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH 1 2 units shifted V , the voltage, at which the E P/E P ratio is 50:50, by 2100 mV. This was paralleled by an approximately two- 0.5 1 2 fold acceleration of the forward rate constant of the E PRE P transition and a similar increase in the rate of steady-state 1 2 cation transport. The temperature dependence of Rb+ uptake yielded an activation energy of ,90 kJ/mol, sugg