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

MERCEDES CENTENO; MARIELA FERREIRA GOMES; ROSSI JUAN PABLO; MONICA MONTES; ROLANDO ROSSI

Tucumán

Congreso; Reunión Anual Sociedad Argentina de Biofisica; 2016

Sociedad Argentina de Biofisica

The Na,K-ATPase is a membrane-bound ion pump that generates electrochemicalgradients for Na+ and K+ across plasma membranes of animalcells. The enzyme oscillates between two major conformations, E1and E2. Under physiological conditions, E1 binds Na+, ATP and Mg2+and forms the phosphorylated state E1P containing occluded Na+, withthe release of ADP. After a conformational transition to E2P, Na+ isreleased and K+ binds and becomes occluded. The subsequent releaseof K+ leads to E1 and the cycle begins again.In this work we report results on the effects of oligomycin, an antibioticfrom Streptomyces diastatochromogenes and epigallocatechin-3--gallate (EGCg), a polyphenolic compound obtained from green tea, asinhibitory agents to isolate the intermediates involved in the transportof Na+. It is proposed that, like oligomycin, EGCc inhibits the enzymeactivity by stabilizing the E1 intermediates, thus blocking the E1P ?E2P conformational change. We therefore evaluated the effects of theseinhibitors on the ATPase activity, the phosphoenzyme level, and thebinding/occlusion of Na+ in the enzyme.Results show that: (i) EGCg inhibited the ATPase activity with a K0:5of 1 mM as previously reported, (ii) the phosphoenzyme level (measuredusing [􀀀32P]ATP) that remains after the addition of K+ washigher in the presence of EGCg than in its absence, and (iii) in the presenceof either oligomycin or EGCg the amount of tightly-bound 22Na+was 18 times higher than with the enzyme thermally inactivated, and theamount of 22Na+ in the enzyme without inhibitors was 8 times higherthan with the inactivated enzyme.These results suggest that, like oligomycin, EGCc stabilizes the E1 conformationof the Na,K-ATPase and allows the isolation of the intermediatescontaining occluded Na+.With grants from ANPCYT, CONICET and UBACYT