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

The Na+/K+-ATPase is an integral plasma membrane glycoprotein 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 is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing 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 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, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

Antiproliferative activity of aqueous and polyphenol-rich extracts of Larrea divaricata Cav. on a melanoma cell line.

Most of the deaths from skin cancer are caused by melanoma, a malignancy in which STAT3 plays a crucial role. The inhibition of STAT3 is considered a potential target to induce cell death, tumor regression and metastasis inhibition. The objective of this work was to evaluate the activity of the aqueous extract of Larrea divaricata (Aq), a fraction rich in polyphenols (EA),and the isolated compound quercetin-3-methyl ether (Q3ME) on B16F10 melanoma cells. The effects of Aq, EA and Q3ME were assessed on B16F10 cells by determining the proliferation, viability, apoptosis induction and the expression and phosphorylation of STAT3. The phytochemical composition of the extracts was determined by High Performance Liquid Chromatography. Aq, EA and Q3ME presented antiproliferative activity on B6F10 cells through p-STAT3 inhibition and early and late apoptosis induction (EC50 EA= ≤0.1 µg/ml; Aq= 316 ± 30 µg/ml; Q3ME= <0.1 µg/ml). L. divaricata could be considered for the development of adjuvant phytotherapies in melanoma treatment.