Glutamate 329 located in the fourth transmembrane segment of the alpha-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions.
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 the 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. Functional analysis on isolated plasma membranes demonstrated that the Glu329-->Gln variant was able to catalyze Na(+)- and K(+)-activated ATPase activity with a maximum turnover number similar if not identical to that of the wild type, but the variant exhibited a reduced affinity for both cations corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold increase in K0.5 for K+. Moreover, the apparent affinity for ATP was increased 15-fold in the variant relative to wild-type Na+,K(+)-ATPase. The Na+,K(+)-ATPase activity of the variant displayed an anomalous pH dependence with a down-shift of the pH optimum and a nearly constant rate in the range between pH 7.0 and 8.7.
Vilsen
B
B
Danish Biomembrane Research Centre, University of Aarhus, Denmark.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D005971:Glutamates / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014162:Transfection
D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium