Amino acid residues of the Na,K-ATPase involved in ouabain sensitivity do not bind the sugar moiety of cardiac glycosides.
The identification of amino acid substitutions in the alpha subunit of the Na,K-ATPase that alter cardiac glycoside sensitivity provides a unique opportunity to examine the role these residues play in binding to the structural domains of these drugs. Substitution of a residue(s) involved in binding to the sugar moiety of cardiac glycosides would be expected to yield similar affinities for ouabain and its aglycone, ouabagenin. Sheep Na,K-ATPase alpha 1 subunit amino acid substitutions previously shown to influence ouabain sensitivity were tested for activity in the presence of ouabain and ouabagenin. These substitutions included both transmembrane and extracellular regions: C104F, D121E, N122D, Q111K, N122K, and Q111R, A112S. Na,K-ATPase activity versus drug concentration curves yielded I50 values over a 1000-fold range for wild type HeLa and HeLa transfectants. Interestingly, the I50 ratio for ouabagenin to ouabain in all mutants tested indicated a 19-24-fold lower affinity of the Na,K-ATPase for ouabagenin. This constant ratio among all mutations tested implies that the first transmembrane region and the first extracellular loop (H1-H2) do not participate in the binding of the sugar moiety of cardiac glycosides.
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
W J
WJ
O'Brien
E T
ET
Wallick
J B
JB
Lingrel
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000031:analogs & derivatives* / Q000494:pharmacology*; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D046911:Macromolecular Substances; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; C008721:ouabagenin