Sites involved in the spontaneous occlusion of K+ in the Na,K-ATPase.

R. M. GONZÁLEZ-LEBRERO; S. B. KAUFMAN; P. J. GARRAHAN; R. C. ROSSI

San Carlos de Bariloche, Río Negro, Argentina

Congreso; Reunión internacional conjunta: XXXII reunión anual de la Sociedad Argentina de Biofísica (SAB), XXXIX reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB) y Bariloche Protein Symposium; 2003

Sociedad Argentina de Biofísica, y Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

The Na+/K+-ATPase is a protein of the cell membrane that exports 3 Na+ and imports 2 K+ ions in each transport cycle, during which these ions become temporarily occluded (trapped within the protein). Occluded K+ can also be formed by mixing the enzyme with the cation in media lacking Na+, Mg2+ and ATP (?direct route?). While K+ occlusion in physiological conditions occurs through extracellular sites of the enzyme, it is believed that occlusion via the direct route takes place through intracellular sites, but this is still unclear. We here investigated whether the sites of the enzyme implicated in the occlusion of K+ via the direct route are extracelullar or intracellular. Under these conditions, K+ occlusion and deocclusion occur through ordered processes with a fast and a slow components of similar sizes. This behaviour resembles that found for K+ deocclusion in the presence of Mg2+ and orthophosphate (MgPi) where the sites involved are extracellular. Using 86Rb+ as a K+ congener, and a double incubation sequence (with 86Rb+ or Rb+) we labelled the slow- or fast-exchange pools. Depending on whether the deocclusion velocity of 86Rb+ in the presence of MgPi was slow or fast after the double incubation sequence, we could infer if the occlusion via the direct route occurred through extracelullar or intracellular sites of the enzyme. Results suggest that the release of Rb+ in media with MgPi take place through the same sites as those from which the cation entered the enzyme, i.e. the external ones.