Characterization of the transition between the direct and physiological routes of occlusion of K+ in the Na+/K+-ATPase caused by addition of Na+.

JOSE LUIS E. MONTI; PABLO G. SCHVARTZ; RODOLFO M. GONZÁLEZ-LEBRERO; SERGIO B. KAUFMAN; PATRICIO J. GARRAHAN; ROLANDO C. ROSSI

Woods Hole, MA, USA.

Congreso; 11th International ATPase Conference & 59th Annual Meeting and Symposium of the Society of General Physiologists.; 2005

Society of General Physiologists.

K+-occluded intermediates of the Na+/K+-ATPase can be obtained either by the direct route, without formation of phosphoenzyme, or by the physiological route, after K+-stimulated dephosphorylation of the phosphoenzyme. Since Na+ is required for phosphorylation by ATP, it is possible to characterize the transition between these two ways of occlusion caused by addition of Na+. With this aim we performed experiments at 25C using a purified preparation of pig kidney Na+/K+-ATPase in media containing 2.5mM ATP, 0.5mM free Mg2+, imidazole-HCl 25mM, pH=7.4, and different concentrations of [86Rb+]RbCl (instead of KCl), NaCl, and CholineCl in order to keep constant the ionic strenght (the total concentration of the last three salts was always 170mM). Reaction time was 6-10 seconds. Results show that, at fixed [Rb+] and for increasing [Na+], occluded Rb+ first increased along a sigmoidal curve and then decreased along a hyperbola to a nonzero value. When [Rb+] increases, the K05 of both the increasing and decreasing phases increases. In the absence of Na+, occluded Rb+ increases with [Rb+] along a hyperbola with a small but significant value as [Rb+] tends to zero (about 2.5% of the maximal amount of occluded Rb+), that could represent a high-affinity component. A model based on the Albers-Post scheme where three Na+ must bind to E1 to prevent occlusion by the direct route and to produce phosphorylation, and where Na+ and K+ compete for the cation binding sites in E1 and in E2P, approximately predicts the results. However, this model fails to predict that occluded Rb+ should decrease to a nonzero value as Na+ concentration increases to infinity. This discrepancy could be partially mimiked by considering that choline can occupy the cation binding sites in E2P and the experimental condition that [choline] decreases when [Na+] increases in order to keep the ionic strenght constant.  [Supported by CONICET, Agencia Nacional de Promoción Científica y Tecnológica and Universidad de Buenos Aires]