Steps involved in the transport of Na+ by the Na,K-ATPase

FERREIRA GOMES M. S.; ROSSI R. C.; CENTENO M. M.; MONTES M. R.; ROSSI J. P. F. C.

San Miguel de Tucumán

Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) ? IX IberoAmerican Congress of Biophysics ? XLV Reunion Anual SAB 2016; 2016

Sociedad Argentina de Biofísica

The Na,K-ATPase is a membrane-bound ion pump that generates electrochemical gradients for Na+ and K+ across plasma membranes of animal cells. The enzyme oscillates between two major conformations, E1 and E2. Under physiological conditions, E1 binds Na+, ATP and Mg2+ and forms the phosphorylated state E1P containing occluded Na+, with the release of ADP. After a conformational transition to E2P, Na+ is released and K+ binds and becomes occluded. The subsequent release of K+ leads to E1 and the cycle begins again. In this work we report results on the effects of oligomycin, an antibiotic from Streptomyces diastatochromogenes and epigallocatechin-3-gallate (EGCg), a polyphenolic compound obtained from green tea, as inhibitory agents to isolate the intermediates involved in the transport of Na+. It is proposed that, like oligomycin, EGCc inhibits the enzyme activity by stabilizing the E1 intermediates, thus blocking the E1P ? E2P conformational change. We therefore evaluated the effects of these inhibitors on the ATPase activity, the phosphoenzyme level, and the binding/occlusion of Na+ in the enzyme. Results show that: (i) EGCg inhibited the ATPase activity with a K0:5 of 1 mM as previously reported, (ii) the phosphoenzyme level (measured using [gamma-32P]ATP) that remains after the addition of K+ was higher in the presence of EGCg than in its absence, and (iii) in the presence of either oligomycin or EGCg the amount of tightly-bound 22Na+ was 18 times higher than with the enzyme thermally inactivated, and the amount of 22Na+ in the enzyme without inhibitors was 8 times higher than with the inactivated enzyme. These results suggest that, like oligomycin, EGCc stabilizes the E1 conformation of the Na,K-ATPase and allows the isolation of the intermediates containing occluded Na+.