Role of homologous ASP334 and GLU319 in human non-gastric H,K- and Na,K-ATPases in cardiac glycoside binding

Biochem Biophys Res Commun. 2007 Apr 27;356(1):142-6. doi: 10.1016/j.bbrc.2007.02.119. Epub 2007 Mar 1.

Abstract

Cardiac steroids inhibit Na,K-ATPase and the related non-gastric H,K-ATPase, while they do not interact with gastric H,K-ATPase. Introducing an arginine, the residue present in the gastric H,K-ATPase, in the second extracellular loop at the corresponding position 334 in the human non-gastric H,K-ATPase (D334R mutation) rendered it completely resistant to 2mM ouabain. The corresponding mutation (E319R) in alpha1 Na,K-ATPase produced a approximately 2-fold increase of the ouabain IC(50) in the ouabain-resistant rat alpha1 Na,K-ATPase and a large decrease of the ouabain affinity of human alpha1 Na,K-ATPase, on the other hand this mutation had no effect on the affinity for the aglycone ouabagenin. These results provide a strong support for the orientation of ouabain in its biding site with its sugar moiety interacting directly with the second extracellular loop.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution*
  • Animals
  • Aspartic Acid / genetics
  • Binding, Competitive / drug effects
  • Biological Transport / drug effects
  • Cardiac Glycosides / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Female
  • Glutamic Acid / genetics
  • H(+)-K(+)-Exchanging ATPase / genetics
  • H(+)-K(+)-Exchanging ATPase / metabolism*
  • Humans
  • Membrane Potentials / drug effects
  • Mutation
  • Oocytes / drug effects
  • Oocytes / metabolism
  • Oocytes / physiology
  • Ouabain / analogs & derivatives
  • Ouabain / pharmacology
  • Protein Subunits / antagonists & inhibitors
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Proton Pump Inhibitors
  • Rabbits
  • Rats
  • Rubidium Radioisotopes / pharmacokinetics
  • Sequence Homology, Amino Acid
  • Sodium-Potassium-Exchanging ATPase / antagonists & inhibitors
  • Sodium-Potassium-Exchanging ATPase / genetics
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Xenopus laevis

Substances

  • Cardiac Glycosides
  • Enzyme Inhibitors
  • Protein Subunits
  • Proton Pump Inhibitors
  • Rubidium Radioisotopes
  • Aspartic Acid
  • Glutamic Acid
  • Ouabain
  • H(+)-K(+)-Exchanging ATPase
  • Sodium-Potassium-Exchanging ATPase
  • ouabagenin