A GLU(329)-] GLN VARIANT OF THE ALPHA-SUBUNIT OF THE RAT-KIDNEY NA-ATPASE CAN SUSTAIN ACTIVE-TRANSPORT OF NA+ AND K+ AND NA+,K+-ACTIVATED ATP HYDROLYSIS WITH NORMAL TURNOVER NUMBER(,K+)

An allelic variant of the ouabain-insensitive rat kidney Na+,K+-ATPase alpha1-isoform was identified by chance in a cDNA library. The varian t differed from the wild-type rat kidney Na+,K+-ATPase by a single G-t o-C base substitution in the cDNA, which on amino acid level gave rise to a glutamine in place of the glutamate residue Glu329 previously su ggested as a likely donator of oxygen ligands for Na+ and K+ binding. The variant cDNA was transfected into COS-1 cells and the transfectant s 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 c ation transport mediated by the ouabain-insensitive Glu329-->Gln varia nt, whose cDNA was shown by polymerase chain reaction amplification to be stably integrated into the COS-I 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 an d wild-type enzyme, and it was thereby demonstrated that the variant h ad a turnover number similar if not identical to that of the wild-type .