The missing link of the Albers-Post model: measurement of Na+-occluded states in fully functional Na+/K+-ATPase

MARIELA S. FERREIRA-GOMES; MERCEDES CENTENO; JUAN PABLO F.C. ROSSI; WANDA VALSECCHI; ELINA MALÉN SAINT MARTIN; NATALYA U. FEDOSOVA; SANTIAGO E. FARAJ; NICOLE CERF; JOSÉ LUIS E. MONTI; ROLANDO C. ROSSI

Divinopolis

Congreso; V Meeting on Cardiotonic Steroids and the Na+-Pump; 2019

Universidade Federal de São João del-Rei

One of the less studied aspects of the Na+/K+-ATPase reaction cycle is the kinetics of formation and breakdown of the intermediates involved in the transport of Na+. According to the Albers-Post model, binding of 3 intracellular Na+ to the enzyme triggers phosphorylation by ATP in the presence of Mg2+ and Na+ becomes occluded in the phosphorylated intermediate E1P. Unlike occlusion of K+ and its congeners, which has been characterized quite extensively in steady-state and pre-steady-state experiments, there are no reports of direct measurements of Na+ occlusion in fully functional preparations of Na+/K+-ATPase. It has only been possible to measure occlusion of Na+ in phosphorylated enzyme pretreated with N-ethylmaleimide or alpha-chymotrypsin, or using CrATP, and in dephosphorylated enzyme in the presence of oligomycin. In any of these conditions, the activity of the enzyme is totally or almost totally inhibited, likely blocked in the E1P->E2P transition. In this work, we present direct evidence of Na+ occlusion in a fully functional preparation of Na+/K+-ATPase. For this purpose, we used epigallocatechin-3-gallate (EGCg) as a quenching reagent combined with a rapid filtration method, thus avoiding significant loss of Na+ from the occluded states of the pump. It was reported that EGCg inhibits ATPase activity by stabilizing the E1 conformation of the pump. Although the effects of EGCg and oligomycin on the Na+/K+-ATPase activity appear to be similar, the solubility in water at room temperature of EGCg (more than 10 millimolar) is three orders of magnitude higher compared to that of oligomycin. This feature allows reaching high enough EGCg concentrations to act as a quenching reagent. Experiments were carried out at 25 °C in media with imidazole-HCl 25 mM, pH 7.4, using Na,K-ATPase partially purified from pig kidney. Control experiments using eosin-Y were performed to report on the E1->E2 conformational changes associated with the binding of Na+ and Rb+ (here used as a K+ congener). Results show that EGCg favors the formation of occluded states containing tightly-bound Na+, and allows their stabilization and isolation, both in equilibrium and during ATP hydrolysis. The level of tightly-bound Na+ appears to increase with [Na+] along a rectangular hyperbola (K0.5 = 3.5 mM; maximum stoichiometry of ca. 3 Na+/enzyme unit). Although the shape of the curve might correspond to three identical and independent sites for Na+, an inspection of the results at very low [Na+] reveals the existence of significant positive interactions between these sites. The value of K0.5 for Na+ obtained from occlusion experiments contrasts with the sub-millimolar value obtained from eosin fluorescence experiments. This apparent discrepancy can be solved considering that, while the binding of a single Na+ is sufficient for inducing the E2-> E1 conformational change, occlusion requires the simultaneous binding of three Na+ ions to the Na+/K+-ATPase. This study uses the reversible inhibition produced by EGCg on Na+/K+-ATPase activity as a tool for kinetic measurements of Na+ transport intermediates. The results are the first published evidence on the occlusion of Na+ in a fully functional preparation of Na+/K+-ATPase and confirm the existence of occluded intermediates during Na+ transport. Furthermore, they allow testing the hypothesis about the mechanism by which the Na+ is bound and transported by the pump, proposed by other authors from studies of the crystalline structure.