The role of Na+ as a substitute for protons in the gastric H,K-ATPase

NICOLE CERF; WANDA VALSECCHI; ROLANDO C. ROSSI; SANTIAGO E. FARAJ; MÓNICA R. MONTES

La Plata, Buenos Aires

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

The gastric H,K-ATPase is responsible for the ATP-dependent exchange of intracellular protons for extracellular potassium ions. The reaction cycle is proposed to be similar to that of the Albers-Post model already used to describe P2-ATPases but the H+/ATP stoichiometry is still under discussion. Due to its high structural similarity with the Na,K-ATPase and the fact that protons can replace sodium as well as potassium in the reaction mechanism of the Na,K-ATPase, the role of sodium as congeners of protons has been evaluated in the H,K-ATPase. Durr et al. (1) presented arguments in favor of Na+ as surrogates for protons regarding the E2P→E1P transition and reducing the apparent affinity for Rb+ whereas Swartz et al. (2) proposed that Na+ acts as a K+ analog increasing the rate of dephosphorylation reaction in the gastric pump.With the aim of using Na+ for measurements of stoichiometry, we analyzed the effects of the cation on conformational transitions, ATPase activity, and cation binding. Experiments were carried out at 25 °C in media with imidazole-HCl 25 mM, pH 7.4, using pig gastric H,K-ATPase-enriched membrane vesicles permeabilized with alameticin (3).Results showed that Na+ increases the eosin fluorescence signal probably by shifting the E1↔E2 equilibrium to the E1 conformation, as it is proposed for H+. A similar effect was observed when increasing the ionic strength, using choline chloride, which raises the question of whether it is specific for Na+. Epigallocatechin-3-gallate inhibits the H,K-ATPase activity but, unlike what we found in the Na,K-ATPase, it failed to increase the affinity for Na+ binding and to favor the occlusion of this cation.