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

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

La Plata

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

Sociedad Argentina de Biofisica

The gastric H,K-ATPase is responsible for the ATP-dependent exchange ofintracellular protons for extracellular potassium ions. The reaction cycle isproposed to be similar to that of the Albers-Post model already used to describeP2-ATPases but the H+/ATP stoichiometry is still under discussion. Due to its highstructural similarity with the Na,K-ATPase and the fact that protons can replacesodium as well as potassium in the reaction mechanism of the Na,K-ATPase, therole of sodium as congeners of protons has been evaluated in the H,K-ATPase. Durret al. (1) presented arguments in favor of Na+ as surrogates for protons regardingthe E2P→E1P transition and reducing the apparent affinity for Rb+ whereas Swartzet al. (2) proposed that Na+ acts as a K + analog increasing the rate ofdephosphorylation reaction in the gastric pump.With the aim of using Na+ for measurements of stoichiometry, we analyzed theeffects of the cation on conformational transitions, ATPase activity, and cationbinding. Experiments were carried out at 25 °C in media with imidazole-HCl 25mM, pH 7.4, using pig gastric H,K-ATPase-enriched membrane vesiclespermeabilized with alameticin (3).Results showed that Na+ increases the eosin fluorescence signal probably byshifting the E1↔E2 equilibrium to the E1 conformation, as it is proposed for H+. Asimilar effect was observed when increasing the ionic strength, using cholinechloride, which raises the question of whether it is specific for Na+.Epigallocatechin-3-gallate inhibits the H,K-ATPase activity but, unlike what wefound in the Na,K-ATPase, it failed to increase the affinity for Na+ binding and tofavor the occlusion of this cation.