Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+‐ATPase provide novel insights into the mechanism of sodium transport

FARAJ, SANTIAGO; VALSECCHI WADA M; FERREIRA GOMES, MARIELA; FEDOSOVA, N; JUAN PABLO ROSSI; MONTES, MÓNICA R.; ROLANDO C ROSSI

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

The Na+/K+-ATPase is an integral glycoprotein of the plasma membrane of all animal cellsthat couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP.The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes thephysical basis of a series of fundamental biological phenomena. The pumping mechanisminvolves the presence of gates alternatively exposing Na+/K+‐ATPase transport sites to theintracellular and extracellular sides and includes the existence of states in which bothgates are simultaneously closed, to wit, occluded states.Unlike for K+, information is lacking about Na+-occluded states, which have only beendetected in species unable to perform a catalytic cycle, including two Na+-containingcrystallographic structures. The current knowledge is that intracellular Na+ must bind tothe transport sites and become occluded upon phosphorylation by ATP to be transportedto the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate(EGCg) to instantaneously stabilize native Na+-occluded intermediates, we isolatedspecies with tightly-bound Na+ in an enzyme able to perform a catalytic cycle, consistentwith a genuine occluded state.We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form ofthe Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, theaddition of ATP does not produce an increase in Na+ occlusion as it would have beenexpected; on the contrary, occluded Na+ transiently decreases while ATP lasts. Theseresults reveal new properties of E1 intermediates of the Albers-Post model for explainingthe Na+ transport pathway.AcknowldegmentsThis work was supported by CONICET, ANPCyT and Universidad de Buenos Aires.