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

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 Met 119 and Ser 124 in the M1–M2 extracellular loop of the 2 isoform by the corresponding Thr 119 and Gln 124 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 ouabainsensitive 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. The ubiquitous Na,K-ATPase, which is responsible for the maintenance of the Na and K gradients in animal cells, functions as the pharmacological receptor for cardiac glycosides. Compounds such as digoxin, digitoxin, and ouabain are plant-derived steroids that bind to Na,K-ATPase with high selectivity and inhibit its transport activity. In myocardial cells, this inhibition results in a sequential increase in intracellular sodium and calcium concentrations and, consequently, an increase in the force of contraction. Especially digoxin is still widely used as an inotropic drug in the treatment of congestive heart failure despite its low therapeutic index. A better understanding of the structural features that determine cardiac glycoside interaction with Na,K-ATPase should help to develop inotropic drugs with better therapeutic effects and lower toxic effects. The Na,K-ATPase is composed of a catalytic -subunit