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+)

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.