GLUTAMATE-329 LOCATED IN THE 4TH TRANSMEMBRANE SEGMENT OF THE ALPHA-SUBUNIT OF THE RAT-KIDNEY NA-ATPASE IS NOT AN ESSENTIAL RESIDUE FOR ACTIVE-TRANSPORT OF SODIUM AND POTASSIUM-IONS(,K+)
B. Vilsen, GLUTAMATE-329 LOCATED IN THE 4TH TRANSMEMBRANE SEGMENT OF THE ALPHA-SUBUNIT OF THE RAT-KIDNEY NA-ATPASE IS NOT AN ESSENTIAL RESIDUE FOR ACTIVE-TRANSPORT OF SODIUM AND POTASSIUM-IONS(,K+), Biochemistry, 32(48), 1993, pp. 13340-13349
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 the amino acid level gave
rise to a glutamine in place of the glutamate residue Glu329, previous
ly suggested as a likely donator of oxygen ligands for Na+ and K+ bind
ing. The variant cDNA was transfected into COS-1 cells and the transfe
ctants 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+-A
TPase. Under these conditions, the viability of the cells depended on
the cation transport mediated by the ouabain-insensitive Glu329 --> Gl
n variant, whose cDNA was shown by polymerase chain reaction amplifica
tion to be stably integrated into the COS-1 cell genome. Functional an
alysis on isolated plasma membranes demonstrated that the Glu329 --> G
ln 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 cati
ons corresponding to a 2-fold increase in K0.5 for Na+ and a 6-fold in
crease in K0.5 for K+. Moreover, the apparent affinity for ATP was inc
reased 15-fold in the variant relative to wild-type Na+,K+-ATPase. The
Na+,K+-ATPase activity of the variant displayed an anomalous pH depen
dence with a down-shift of the pH optimum and a nearly constant rate i
n the range between pH 7.0 and 8.7.