The third sodium binding site of Na,K-ATPase is functionally linked to acidic pH-activated inward current

Sodium- and potassium-activated adenosine triphosphatases (Na,K-ATPase) is the ubiquitous active transport system that maintains the Na+ and K+ gradients across the plasma membrane by exchanging three intracellular Na+ ions against two extracellular K+ ions. In addition to the two cation binding sites homologous to the calcium site of sarcoplasmic and endoplasmic reticulum calcium ATPase and which are alternatively occupied by Na+ and K+ ions, a third Na+-specific site is located close to transmembrane domains 5, 6 and 9, and mutations close to this site induce marked alterations of the voltage-dependent release of Na+ to the extracellular side. In the absence of extracellular Na+ and K+, Na,K-ATPase carries an acidic pH-activated, ouabain-sensitive "leak" current. We investigated the relationship between the third Na+ binding site and the pH-activated current. The decrease (in E961A, T814A and Y778F mutants) or the increase (in G813A mutant) of the voltage-dependent extracellular Na+ affinity was paralleled by a decrease or an increase in the pH-activated current, respectively. Moreover, replacing E961 with oxygen-containing side chain residues such as glutamine or aspartate had little effect on the voltage-dependent affinity for extracellular Na+ and produced only small effects on the pH-activated current. Our results suggest that extracellular protons and Na+ ions share a high field access channel between the extracellular solution and the third Na+ binding site.