title: The third sodium binding site of Na,K-ATPase is functionally linked to acidic pH-activated inward current.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Lausanne, CH-1005 Lausanne, Switzerland.
authors.forename: Ciming
authors.initials: C
authors.lastname: Li
authors.forename: Käthi
authors.initials: K
authors.lastname: Geering
authors.forename: Jean-Daniel
authors.initials: JD
authors.lastname: Horisberger
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008564:Membrane Potentials; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
chemical_list: D011994:Recombinant Proteins; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium