B. Vilsen, FUNCTIONAL CONSEQUENCES OF MUTATION ASN(326)-]LEU IN THE 4TH TRANSMEMBRANE SEGMENT OF THE ALPHA-SUBUNIT OF THE RAT-KIDNEY NA-ATPASE(,K+), FEBS letters, 363(1-2), 1995, pp. 179-183
Site-specific mutagenesis was used to replace Asn(326) in transmembran
e segment M4 of the ouabain-insensitive alpha 1-isoform of rat kidney
Na+,K+-ATPase, Mutant Asn(326) --> Leu was functional as demonstrated
by the ability of COS cells expressing the mutant enzyme to grow in th
e presence of ouabain. In three independent assays encompassing Na+ ti
trations of Na+,K+-ATPase activity, Na+-ATPase activity, and phosphory
lation from ATP, the Asn(326) --> Leu mutant displayed a reduced appar
ent affinity for Na+. By contrast, this mutant exhibited a slightly in
creased apparent affinity for K+ relative to the wild-type enzyme. In
the presence of Na+ without K+, the Asn(326) --> Leu mutant hydrolyzed
ATP at a high rate corresponding to 32% of the maximal Na+,K+-ATPase
activity, and the rate of dephosphorylation of the phosphoenzyme inter
mediate was enhanced in the mutant relative to that of the wild-type e
nzyme. Oligomycin, known to stabilize the Na+-occluded phosphoenzyme i
ntermediate, reduced the dephosphorylation rate of the mutant and incr
eased the steady-state phosphoenzyme level formed by the mutant at lea
st 3-fold, whereas an increase in the steady-state phosphoenzyme level
of only 10-15% was determined for the mild-type enzyme. The molecular
turnover number for the Na+,K+-ATPase reaction, calculated when the s
teady-state phosphoenzyme level obtained in the presence of oligomycin
was taken as a measure of the concentration of active sites, was slig
htly reduced relative to that of the wild-type enzyme. The data are di
scussed in terms of a role for Asn(326) in binding of cytoplasmic Naand in mediation of inhibition of dephosphorylation.