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

J Membr Biol. 2006;213(1):1-9. doi: 10.1007/s00232-006-0035-0. Epub 2007 Mar 8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Female
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Membrane Potentials
  • Models, Biological
  • Mutagenesis, Site-Directed
  • Oocytes / metabolism
  • Ouabain / pharmacology
  • Potassium / metabolism
  • Protein Structure, Tertiary
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sodium / metabolism
  • Sodium-Potassium-Exchanging ATPase / chemistry
  • Sodium-Potassium-Exchanging ATPase / genetics
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Xenopus

Substances

  • Recombinant Proteins
  • Ouabain
  • Sodium
  • Sodium-Potassium-Exchanging ATPase
  • Potassium