Functional Expression of Na⁺/K⁺-ATPase α and β Isoforms

Abstract

Both the catalytic (a) and glycosylated (β) subunits of the Na+/K+-ATPase are encoded by a family of genes. Three Na⁺/K⁺-ATPase a isoforms (α1, α2 and α3) and two ß (ß1 and ß2) have been identified in mammals (3). The differential properties of the α and ß polypeptides in tissue expression, development and hormonal stimulation, suggest that each isoform might be playing a specific physiological role. The study of the properties of the Na⁺/K+-ATPase isoforms has been complicated by their coexistence in different tissues and because of the difficulty that their isolation represents. To gain insight into the functional characteristics of the Na⁺/K⁺-ATPase isoforms, we have successfully used the baculovirus expression system to express the rodent al, a2 and a3 Na+/K+-ATPase isoforms, separately and in combination with ß1 in Sf-9 insect cells (1). These cells have low levels of endogenous Na⁺/K⁺-ATPase and are able to express high amounts of the baculovirus induced Na⁺/K⁺-ATPase polypeptides. The coexpression of α1, α2 and α3 with ß1 resulted in the production of catalytically competent Na⁺/K⁺-ATPase molecules as determined by ouabain-sensitive ATPase activity, ouabain and K-sensitive ATP phosphorylation and [³H]ouabain binding. a2ß1 and α3ß1 isozymes display a high sensitivity to ouabain, with α3ß1 the more sensitive. At present, the different α and ß isoform combinations that result in active Na⁺/K+-ATPase enzyme have not been completely determined. Here we show coexpression of the α1, α2 and α3 isoforms in combination with the ß2 subunit. All α isoforms are able to associate with the ß2 subunit in virally infected insect cells and the Na⁺/K⁺ -ATPase isozymes produced are catalytically active.