Measurement of Na+-occluded States in the Na,K-ATPase During its Normal Functioning

SANTIAGO FARAJ; WANDA VALSECHI; MALÉN SAINT MARTIN; MARIELA FERREIRA GOMES; JUAN PABLO ROSSI; MONTES MÓNICA; ROSSI ROLANDO

La Plata

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

Sociedad Argentina de Biofisica

According to the Albers-Post model, binding of 3 intracellular Na+ to the E1 state ofthe 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 theextracellular medium after the E1P-->E2P conformational transition. Na+ occlusionhas never been reported in unmodified enzyme; therefore, there is no informationon the kinetics of Na+-transport intermediates during the normal functioning of theNa,K-ATPase. Here, we present the first results of Na+ occlusion during thehydrolysis 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+, thereaction media were injected into a chamber with a Millipore filter through whichan ice-cold washing solution was flowing. The solution contained 1 mMepigallocatechin-3-gallate to stabilize the Na+-occluded states. Formation of theE2P 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 aprox. 3 Na+/enzyme unit); (ii) in the presence of 0.5 mM Mg2+ thecurve becomes sigmoidal (K0.5= 7.2 mM; max. stoichiometry aprox. 3 Na+/enzymeunit); and (iii) decreases with [Mg2+] and with [Rb+]. The time course of tightlyboundNa+ after addition of 5 or 15 mM ATP and 0.5 mM Mg2+ showed a transientdecrease, which lasted the more the higher the concentration of ATP. Experimentsusing RH421 suggest that these time courses reflect the formation and breakdownof phosphorylated intermediates until ATP is totally hydrolyzed. Our results aresolid evidence that the tightly-bound Na+ remains occluded in the Na,K-ATPase andthat we are measuring the occlusion of Na+ during ATP hydrolysis.AcknowldegmentsThis project was supported with grants from Consejo Nacional de Investigaciones Científicas y Técnicas,Universidad de Buenos Aires, and Agencia Nacional de Promoción Científica y Tecnológica, Argentina.