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New Molecular Determinants Controlling the Accessibility of Ouabain to Its Binding Site in Human Na,K-ATPase Isoforms

Institute of Pharmacology and Toxicology of the University, Lausanne, Switzerland

Inhibition of Na,K-ATPase 2 isoforms in the human heart is supposed to be involved in the inotropic effect of cardiac glycosides, whereas inhibition of 1 isoforms may be responsible for their toxic effects. Human Na,K-ATPase 1 and 2 isoforms exhibit a high ouabain affinity but significantly differ in the ouabain association and dissociation rates. To identify the structural determinants that are involved in these differences, we have prepared chimeras between human 1 and 2 isoforms and 2 mutants in which nonconserved amino acids were exchanged with those of the 1 isoform, expressed these constructs in Xenopus laevis oocytes, and measured their ouabain binding kinetics. Our results show that replacement of Met¹¹⁹ and Ser¹²⁴ in the M1–M2 extracellular loop of the 2 isoform by the corresponding Thr¹¹⁹ and Gln¹²⁴ of the 1 isoform shifts both the fast ouabain association and dissociation rates of the 2 isoform to the slow ouabain binding kinetics of the 1 isoform. The amino acids at position 119 and 124 cooperate with the M7–M8 hairpin and are also responsible for the small differences in the ouabain affinity of the ouabain-sensitive 1 and 2 isoforms. Thus, we have identified new structural determinants in the Na,K-ATPase -subunit that are involved in ouabain binding and probably control, in an isoform-specific way, the access and release of ouabain to and from its binding site.

Address correspondence to: Käthi Geering, Institute of Pharmacology et Toxicology of the University, rue du Bugnon 27, CH-1005 Lausanne, Switzerland. E-mail: kaethi.geering{at}ipharm.unil.ch

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