COMPARISON OF THE SUBSTRATE DEPENDENCE PROPERTIES OF THE RAT NA,K-ATPASE ALPHA-1, ALPHA-2, AND ALPHA-3 ISOFORMS EXPRESSED IN HELA-CELLS

The role of multiple isoforms for the a subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha-subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha-2 and alpha-3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha-2 and Q108R and N119D for alpha-3). cDNAs encoding the rat alpha-1 subunit, which is naturally ouabain-resistant, and rat alpha-2 and alpha-3, which were mutated to ouabain resistance (designated rat alpha-2* and rat alpha-3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1-mu-M ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 congruent-to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha-1, rat alpha-2*, or rat alpha-3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha-1 and rat alpha-2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha-3* (apparent K(Na+) (millimolar): rat alpha-1 = 1.15 +/- 0.13; rat alpha-2* = 1.05 +/- 0.11; rat alpha-3* = 3.08 +/- 0.06). Additionally, rat alpha-3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha-1 or rat alpha-2* (apparent K0.5 (millimolar): rat al = 0.43 +/- 0.12; rat alpha-2* = 0.54 +/- 0.15; rat alpha-3* = 0.21 +/- 0.04) and all three isoforms had similar apparent affinities for K+ (apparent K(K+): rat alpha-1 = 0.45 +/- 0.01; rat alpha-2* = 0.43 +/- 0.004; rat alpha-3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha-isoforms expressed in the same cell type.