Characterization of the fourth α isoform of the Na,K-ATPase

The Na,K-ATPase is a major ion transport protein found in higher eukaryotic cells. The enzyme is composed of two subunits, alpha and beta, and tissue-specific isoforms exist for each of these, alpha 1, alpha 2 and alpha 3 and beta 1, beta 2 and beta 3. We have proposed that an additional alpha isoform, alpha 4, exists based on genomic and cDNA cloning. The mRNA for this gene is expressed in rats and humans, exclusively in the testis, however the expression of a corresponding protein has not been demonstrated. In the current study, the putative alpha 4 isoform has been functionally characterized as a novel isoform of the Na,K-ATPase in both rat testis and in alpha 4 isoform cDNA transfected 3T3 cells. Using an alpha 4 isoform-specific polyclonal antibody, the protein for this novel isoform is detected for the first time in both rat testis and in transfected cell lines. Ouabain binding competition assays reveal the presence of high affinity ouabain receptors in both rat testis and in transfected cell lines that have identical K-D values. Further studies of this high affinity ouabain receptor show that it also has high affinities for both Na+ and K+. The results from these experiments definitively demonstrate the presence of a novel isoform of the Na,K-ATPase in testis.