Role of homologous ASP334 and GLU319 in human non-gastric H,K- and Na,K-ATPases in cardiac glycoside binding.
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.
Radkov
Rossen
R
Department of Physiology, Pharmacology, Metabolism, and Cardiovascular Sciences, University of Toledo College of Medicine, Toledo, OH 43614, USA.
Kharoubi-Hess
Solange
S
Schaer
Danièle
D
Modyanov
Nikolai N
NN
Geering
Käthi
K
Horisberger
Jean-Daniel
JD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002301:Cardiac Glycosides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D018698:Glutamic Acid / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010042:Ouabain / Q000031:analogs & derivatives / Q000494:pharmacology; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054328:Proton Pump Inhibitors; D011817:Rabbits; D051381:Rats; D012414:Rubidium Radioisotopes / Q000493:pharmacokinetics; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D002301:Cardiac Glycosides; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D054328:Proton Pump Inhibitors; D012414:Rubidium Radioisotopes; D001224:Aspartic Acid; D018698:Glutamic Acid; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; C008721:ouabagenin