title: Substitution of transmembrane residues with hydrogen-bonding potential in the alpha subunit of Na,K-ATPase reveals alterations in ouabain sensitivity.
abstract: The role of H-bonding amino acids as determinants of ouabain affinity in the Na,K-ATPase was examined. Site-directed mutagenesis was used to substitute 21 conserved amino acid residues in the sheep alpha-subunit transmembrane regions. The amino acids were changed from residues which possess side chains capable of forming H-bonds with specific cardiac glycoside moieties such as the lactone ring or sugar(s) to residues unable to participate in H-bonding. The effect of each of these amino acid replacements on the affinity of the Na,K-ATPase for ouabain was initially assessed by screening the altered enzymes for the ability to confer ouabain resistance when expressed in otherwise sensitive HeLa cells. Three of the substitutions (Tyr-108 to Ala, Cys-104 to Ala, and Cys-104 to Phe) were able to confer resistance to the normally sensitive HeLa cells. Stable cell lines, each expressing one of the altered enzymes, were further characterized in terms of ouabain-inhibitable cell growth and Na,K-ATPase activity. Cell lines expressing the alpha 1-isoform substitution Y108A, C104A, or C104F contained a Na,K-ATPase activity which gave an I50 value for enzyme inhibition 9-, 6-, and 150-fold greater, respectively, than the endogeneous HeLa or sheep enzyme. These data show that Tyr-108 and Cys-104 of the alpha subunit are determinants of ouabain affinity. Cys-104 has also been shown to be a determinant of ouabain sensitivity in Xenopus laevis [Canessa, C. M., Horisberger, J.-D., Louvard, D., & Rossier, B. C. (1992) EMBO J. 11, 1681-1687].(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
authors.forename: P J
authors.initials: PJ
authors.lastname: Schultheis
authors.forename: J B
authors.initials: JB
authors.lastname: Lingrel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004351:Drug Resistance / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010042:Ouabain / Q000494:pharmacology*; D012313:RNA / Q000235:genetics; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D010042:Ouabain; D012313:RNA; D000254:Sodium-Potassium-Exchanging ATPase