Kinetic analysis of ouabain binding to native and mutated forms of Na,K-ATPase and identification of a new region involved in cardiac glycoside interactions.
Cardiac glycosides inhibit the Na,K-ATPase by binding to the catalytic alpha subunit of the enzyme. Site-directed mutagenesis of the H1-H2 domain has demonstrated the importance of this region in determining cardiac glycoside affinity. In this study, random mutagenesis was used to identify an amino acid, arginine 880, in the COOH-terminal portion of the alpha subunit which influences the sensitivity of the enzyme to ouabain. This residue is predicted to reside in the H7-H8 extracellular loop. Conversion of arginine 880 to a proline causes a 10-fold increase in the dissociation rate constant and a 2-fold increase in the association rate constant for [3H]ouabain binding. This results in an enzyme with a KD for ouabain 5-fold higher than the wild-type sheep alpha 1 isoform. These data are compatible with arginine 880 comprising a portion of the ouabain binding site. Furthermore, if arginine 880 is at the physical binding site, then this finding lends support to models that place this amino acid extracellularly since cardiac glycosides interact with the extracellular surface of the Na,K-ATPase. The ouabain binding characteristics of substitution R880P were compared with those of several different Na,K-ATPases, each of which contains a single amino acid substitution in the H1-H2 region of the alpha subunit. The substituted enzymes, C104A, Y108A, E116Q, P118K, and Y124F, vary considerably in their rates of dissociation (1-4-fold increase in the dissociation rate constant). In addition, the rate of association of [3H]ouabain binding to substitution P118K is 2-fold slower than that of the wild-type enzyme. These results suggest that the H1-H2 domain may participate directly in ouabain binding as well as be involved in conformational changes, both of which could affect the sensitivity of the enzyme to ouabain.
Schultheis
P J
PJ
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Wallick
E T
ET
Lingrel
J B
JB
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002301:Cardiac Glycosides / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010042:Ouabain / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D002301:Cardiac Glycosides; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase