New molecular determinants controlling the accessibility of ouabain to its binding site in human Na,K-ATPase α isoforms

Inhibition of Na,K-ATPase alpha2 isoforms in the human heart is supposed to be involved in the inotropic effect of cardiac glycosides, whereas inhibition of alpha1 isoforms may be responsible for their toxic effects. Human Na, K-ATPase alpha1 and alpha2 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 alpha1 and alpha2 isoforms and alpha2 mutants in which nonconserved amino acids were exchanged with those of the alpha1 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 alpha2 isoform by the corresponding Thr(119) and Gln(124) of the alpha1 isoform shifts both the fast ouabain association and dissociation rates of the alpha2 isoform to the slow ouabain binding kinetics of the alpha1 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 alpha1 and alpha2 isoforms. Thus, we have identified new structural determinants in the Na,K-ATPase alpha-subunit that are involved in ouabain binding and probably control, in an alpha isoform-specific way, the access and release of ouabain to and from its binding site.