Abstract
The role of multiple isoforms for the alpha 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 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal 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 KNa+ (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 alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: 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.
Highlights
Our results suggest that the rat a3* isoform-containinmg ol
We have observed similar manner, the naturally ouabain-sensitive rat a 2 and a 3 en- apparent affinities for K+ among all isoforms and a slightly zymes were converted to ouabain-resistant forms by site- higher apparent affinity for ATP in directed mutagenesis of 2 amino acid residues in the first the rat a3*-containing enzyme compared with rat a1 or a2*. extracellular domain of the protein
Studying the properties of the three isoforms expressed in These results suggested that the a3isoform confers a higher
Summary
FuncCtihoanraalcterization of Na,K-ATPase CY Isoforms ecules exhibit a slightly higher apparent affinity for ATP at of altering some property of rat a 2 or rat a 3 by converting the low affinity site than either rat a1 or rat a2* The means kidney (al) and ouabain-sensitive dog kidney (al) enzymes of the values obtained suggest an approximate 2-fold did not reveal significant differences in any functional paramdifference between the rat a1 and a2* isoforms and the rat eter other than ouabain affinity (39).
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