Ouabain and substrate affinities of human Na+-K+-ATPase α1β1, α2β1, and α3β1 when expressed separately in yeast cells

Human Na+-K+-ATPase alpha (1)beta (1), alpha (2)beta (1), and alpha (3)beta (1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha (1)beta (1) and alpha (3)beta (1) at 37 degreesC and 32 nM for alpha (2)beta (1), indicating that the human alpha -subunit isoforms have a similar high affinity for cardiac glycosides. K-0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows: alpha (2) (6.3 +/-2.4 mM) > alpha (3) (1.6 +/-0.5 mM) approximate to alpha (1) (0.9 +/-0.6 mM), and K-0.5 values for Na+ antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha (1)beta (1) (6,652 min(-1)) was about twice as high as that by alpha (3)beta (1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na+-K(+)ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na+ pumps expressed in Xenopus oocytes, the alpha (2)beta (1) complex in yeast membranes was significantly less stable than alpha (1)beta (1) or alpha (3)beta (1), resulting in a lower functional expression level. The alpha (2)beta (1) complex was also more easily denatured by SDS than was the alpha (1)beta (1) or the alpha (3)beta (1) complex.