Kinetics of the K+-transport steps by the Na+/K+-ATPase.

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

Río de Janeiro, Brasil

Congreso; V Iberoamerican Congress of Biophysics; 2003

Sociedad Brasileña de Biofísica, Sociedad de Biofísicos Latinoamericanos, Sociedad de Biofísica de España

Ion transport through the Na+/K+-ATPase involves steps where the transported cations become transitorily hidden (occluded) into the pumping structure. The normal functioning of the pump implies the following steps: the E1 conformer of the enzyme, in the presence of internal Na+ and Mg2+ and ATP, becomes phosphorylated to E1P, which converts to E2P releasing external Na+. E2P reacts with external K+ and rapidly dephosphorylates to E2, which is converted to E1 with the release of internal K+ (a reaction strongly stimulated by ATP). While Na+ becomes occluded in E1P, K+ becomes occluded in E2. Occlusion of K+ can occur either after binding of the cation to external sites in E2P and promoting the fast dephosphorylation of the enzyme or spontaneously, after mixing the cation with the enzyme in media of low ionic strength, in the absence of Na+, ATP and Mg2+. It is believed that occlusion of K+ under these latter conditions occurs through intracellular sites, representing the reversion of the releasing reaction into the cytoplasm. We here show results strongly indicating that spontaneous occlusion and deocclusion of two K+ involve external, rather than internal sites. The experiments take advantage of the fact that the two K+ become occluded/deoccluded through sequential processes and of evidence that MgPi-promoted deocclusion would occur through extracellular sites.