A Glu329-->Gln variant of the alpha-subunit of the rat kidney Na+,K(+)-ATPase can sustain active transport of Na+ and K+ and Na+,K(+)-activated ATP hydrolysis with normal turnover number

An allelic variant of the ouabain-insensitive rat kidney Na+,K(+)-ATPase alpha 1-isoform was identified by chance in a cDNA library. The variant differed from the wild-type rat kidney Na+,K(+)-ATPase by a single G-to-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously suggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectants expanded with success into stable cell lines that were able to grow in the presence of a concentration of ouabain highly cytotoxic to the parental cells containing only the endogenous COS-1 cell Na+,K(+)-ATPase. Under these conditions, the viability of the cells depended on the cation transport mediated by the ouabain-insensitive Glu329-->Gln variant, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-1 cell genome. The maximum specific ATP hydrolysis activity of isolated plasma membranes of the Glu329-->Gln variant did not differ significantly from that of plasma membranes containing the wild type. A method was established for measurement of the phosphorylation capacity of the expressed Glu329-->Gln variant and wild-type enzyme, and it was thereby demonstrated that the variant had a turnover number similar if not identical to that of the wild-type.