Intracellular Proton Binding is Voltage-Dependent and Rate-Limiting for the Gastric H,K-ATPase Under in vivo Conditions

Compared to the extensively studied Na,K-ATPase reaction cycle, much less is known about the voltage-dependent steps in the pump cycle of gastric H,K-ATPase. Due to the overall electroneutral transport stoichiometry of the proton pump, its voltage-dependent ion transport properties cannot readily be determined by standard electrophysiological techniques.Therefore, we use a combination of two biophysical techniques to investigate H,K-ATPase activity: voltage-clamp-fluorometry on the tetramethylrhodamine-6-maleimide-labeled H,K-ATPase variant αS806C to monitor the voltage-dependent distribution between E1P/E2P-states (Ref. 1) and voltage-controlled Rb+ uptake measurements to assess the steady-state ion transport activity under various pH and ionic conditions in Xenopus oocytes.Both the steady-state E1P/E2P-distribution and Rb+ uptake of the gastric H,K-ATPase are highly sensitive towards changes in intracellular pH (which can be achieved by adding weak organic acids like butyric acid to the extracellular solution), whereas even larger changes in the extracellular pH do neither influence the conformational E1P/E2P-equilibrium nor transport activity. An intracellular acidification of approximately 0.5 pH units results in a large negative shift (∼100 mV) of the voltage-dependent fluorescence amplitudes and an approximately two-fold acceleration of the reciprocal time constants at positive membrane potentials.One possible interpretation of these results is that proton binding takes place in a shallow intracellular ion access channel (apparent well depth: 0.3-0.5).Since maximal rubidium uptake at saturating concentrations is strongly stimulated by intracellular acidification, the voltage-sensitive intracellular proton binding step is apparently rate-limiting for the overall transport activity under physiological conditions. These findings highlight the need for cellullar mechanisms which increase the availability of protons at the cytoplasmic face of the pump, such as CO2-producing mitochondria, sub-membrane carbonic anhydrase and the basolateral Cl-/HCO3- exchanger that are characteristic for parietal cells.References1. Da1/4rr et al. (2009) JBC 284, 20147-54