Measurement of Na+-occluded states in the Na,K-ATPase during its normal functioning

FARAJ SE; VALSECCHI WM; SAINT MARTIN EM; FERREIRA GOMES MS; ROSSI JPFC; MONTES MR; ROSSI RC

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

According to the Albers-Post model, binding of 3 intracellular Na+ to the E1 state of the Na,K-ATPase triggers phosphorylation by ATP in the presence of Mg2+, and Na+ becomes occluded in the phosphorylated intermediate E1P. Na+ is released to the extracellular medium after the E1P-->E2P conformational transition. Na+ occlusion has never been reported in unmodified enzyme; therefore, there is no information on the kinetics of Na+-transport intermediates during the normal functioning of the Na,K-ATPase. Here, we present the first results of Na+ occlusion during the hydrolysis of ATP.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. Bound Na+ was measuredusing 22Na+. To stop the reactions and isolate the species with occluded Na+, the reaction media were injected into a chamber with a Millipore filter through which an ice-cold washing solution was flowing. The solution contained 1 mMepigallocatechin-3-gallate to stabilize the Na+-occluded states. Formation of the E2P intermediate was monitored using the fluorescent probe RH421.Results from equilibrium experiments show that the level of tightly-bound 22Na+:(i) increases with [Na+] along a rectangular hyperbola (K0.5= 3.5 mM; max.stoichiometry approx. 3 Na+/enzyme unit); (ii) in the presence of 0.5 mM Mg2+ the curve becomes sigmoidal (K0.5= 7.2 mM; max. stoichiometry approx. 3 Na+/enzyme unit); and (iii) decreases with [Mg2+] and with [Rb+]. The time course of tightly bound Na+ after addition of 5 or 15 micromolar ATP and 0.5 mM Mg2+ showed a transient decrease, which lasted the more the higher the concentration of ATP. Experiments using RH421 suggest that these time courses reflect the formation and breakdown of phosphorylated intermediates until ATP is totally hydrolyzed. Our results are solid evidence that the tightly-bound Na+ remains occluded in the Na,K-ATPase and that we are measuring the occlusion of Na+ during ATP hydrolysis.