O'Brien W J, Wallick E T, Lingrel J B
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
J Biol Chem. 1993 Apr 15;268(11):7707-12.
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.
钠钾-ATP酶α亚基中改变强心苷敏感性的氨基酸替换的鉴定，为研究这些残基在与这些药物的结构域结合中所起的作用提供了独特的机会。与强心苷糖部分结合的残基的替换，预计会产生对哇巴因及其苷元哇巴因配基相似的亲和力。对先前已证明影响哇巴因敏感性的绵羊钠钾-ATP酶α1亚基氨基酸替换，在存在哇巴因和哇巴因配基的情况下测试其活性。这些替换包括跨膜区和细胞外区：C104F、D121E、N122D、Q111K、N122K、Q111R、A112S。野生型HeLa细胞和HeLa转染细胞的钠钾-ATP酶活性与药物浓度曲线在1000倍的范围内产生了半数抑制浓度（I50）值。有趣的是，在所有测试的突变体中，哇巴因配基与哇巴因的I50比值表明钠钾-ATP酶对哇巴因配基的亲和力低19 - 24倍。所有测试突变中的这个恒定比值意味着第一个跨膜区和第一个细胞外环（H1 - H2）不参与强心苷糖部分的结合。
