Leucine 332 at the boundary between the fourth transmembrane segment and the cytoplasmic domain of Na+,K+-ATPase plays a pivotal role in the ion translocating conformational changes.

Mutants Gly330-->Ala, Leu332-->Ala, Leu332-->Pro, and Pro780-->Ala of the alpha1-isoform of rat kidney Na+,K+-ATPase were expressed in COS-1 cells to active site concentrations between 20 and 70 pmol per mg of membrane protein. The functional properties of the mutants were characterized by titrations of Na+-, K+-, and ATP-dependencies of Na+,K+-ATPase activity as well as by a series of assays measuring the K+-dependence of the steady-state phosphoenzyme level, the kinetics of dephosphorylation under a variety of conditions, and the ADP-ATP exchange activity. In mutants Gly330-->Ala, Leu332-->Ala, and Leu332-->Pro, the molecular turnover number was reduced relative to that of the wild-type Na+,K+-ATPase, and the steady-state phosphoenzyme level was high even in the presence of several millimolar K+. At a low Na+ concentration in the absence of K+, mutants Leu332-->Pro and Gly330-->Ala displayed high ADP-ATP exchange activity and formed a high level of the ADP-sensitive phosphoenzyme (E1P). The phosphoenzyme decayed slowly following a jump in salt concentration and chase with ATP and K+. Hence, the conversion of E1P to the K+-sensitive phosphoenzyme (E2P) was inhibited in mutants Leu332-->Pro and Gly330-->Ala. In the Leu332-->Ala mutant, a high 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 indicated that in the Leu332-->Ala mutant K+ induced reversal of the phosphoenzyme 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.