Abstract

Inhibition of Na,K-ATPase α2 isoforms in the human heart is supposed to be involved in the inotropic effect of cardiac glycosides, whereas inhibition of α1 isoforms may be responsible for their toxic effects. Human Na,K-ATPase α1 and α2 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 α1 and α2 isoforms and α2 mutants in which nonconserved amino acids were exchanged with those of the α1 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 α2 isoform by the corresponding Thr¹¹⁹ and Gln¹²⁴ of the α1 isoform shifts both the fast ouabain association and dissociation rates of the α2 isoform to the slow ouabain binding kinetics of the α1 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 α1 and α2 isoforms. Thus, we have identified new structural determinants in the Na,K-ATPase α-subunit that are involved in ouabain binding and probably control, in an α isoform-specific way, the access and release of ouabain to and from its binding site.