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

FARAJ SE; VALSECCHI WM; FERREIRA-GOMES MS; FEDOSOVA NU; ROSSI JPFC; MONTES MR; ROSSI RC

Rosario, Santa Fé, ARGENTINA

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 cells that 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 the physical basis of a series of fundamental biological phenomena. The pumping mechanism involves the presence of gates alternatively exposing Na+/K+‑ATPase transport sites to the intracellular and extracellular sides and includes the existence of states in which both gates are simultaneously closed, to wit, occluded states. Unlike for K+, information is lacking about Na+-occluded states, which have only been detected in species unable to perform a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate (EGCg) to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly-bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases while ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.