LEUCINE-332 AT THE BOUNDARY BETWEEN THE 4TH TRANSMEMBRANE SEGMENT ANDTHE CYTOPLASMIC DOMAIN OF NA-ATPASE PLAYS A PIVOTAL ROLE IN THE ION TRANSLOCATING CONFORMATIONAL-CHANGES(,K+)
B. Vilsen, LEUCINE-332 AT THE BOUNDARY BETWEEN THE 4TH TRANSMEMBRANE SEGMENT ANDTHE CYTOPLASMIC DOMAIN OF NA-ATPASE PLAYS A PIVOTAL ROLE IN THE ION TRANSLOCATING CONFORMATIONAL-CHANGES(,K+), Biochemistry, 36(43), 1997, pp. 13312-13324
Mutants Gly330-->Ala, Leu332-->Ala, Leu332-->Pro, and Pro780-->Ala of
the alpha(1)-isoform of rat kidney Na+,K+-ATPase were expressed in COS
-l cells to active site concentrations between 20 and 70 pmol per mg o
f membrane protein, The functional properties of the mutants were char
acterized by titrations of Na+-, K+-, and ATP-dependencies of Na+,K+-A
TPase activity as well as by a series of assays measuring the K+-depen
dence of the steady-state phosphoenzyme level, the kinetics of dephosp
horylation under a variety of conditions, and the ADP-ATP exchange act
ivity. In mutants Gly330-->Ala, Leu332-->Ala, and Leu332-->Pro, the mo
lecular turnover number was reduced relative to that of the wildtype N
a+,K+-ATPase, and the steady-state phosphoenzyme level was high even i
n the presence of several millimolar K+ At a low Na+ concentration in
the absence of K+, mutants Leu332-->Pro and Gly330-->Ala displayed hig
h ADP-ATP exchange activity and formed a high level of the ADP-sensiti
ve phosphoenzyme (E1P), The phosphoenzyme decayed slowly following a j
ump in salt concentration and chase with ATP and K+, Hence, the conver
sion of E1P to the K+-sensitive phosphoenzyme (E2P) was inhibited in m
utants Leu332-->Pro and Gly330-->Ala. In the Leu332-->Ala mutant, a hi
gh level of E2P was accumulated in the absence of K+, and the ADP-ATP
exchange activity was low at low Na+ concentration in the absence of K
+, but rose sharply on addition of K+. Dephosphorylation experiments i
ndicated that in the Leu332-->Ala mutant K+ induced reversal of the ph
osphoenzyme interconversion, forming E1P from E2P. Leu332 is therefore
a pivotal residue in the conformational change. Mutants Gly330-->Ala
and Pro780-->Ala displayed reduced K+ affinities relative to the wild
type, determined in three independent assays.