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.
Vilsen
B
B
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002460:Cell Line; D004247:DNA; D018076:DNA, Complementary; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D005973:Glutamine / Q000235:genetics; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D005971:Glutamates; D011994:Recombinant Proteins; D005973:Glutamine; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D004247:DNA; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium