FUNCTIONAL CONSEQUENCES OF AMINO-TERMINAL DIVERSITY OF THE CATALYTIC SUBUNIT OF THE NA,K-ATPASE

One region of marked sequence diversity among the highly homologous a isoforms of the Na,K-ATPase is the lysine rich NH2 terminus. Expressio n of a mutant cDNA encoding an alpha 1 protein, minus the 32 NH2-termi nal residues, results in a modified enzyme (alpha 1M32), which be have s similarly to alpha 1 in overall Na/K exchange activity (V-max) and a pparent affinities for intracellular Na+ and extracellular K+. However , with membranes isolated from HeLa cells expressing the rat alpha 1M3 2 mutant, as well as membranes from cells expressing the rat alpha 1 a nd the ouabain-resistant mutated forms of rat alpha 2 (alpha 2) and a lpha 3 (alpha 3) developed by Jewell and Lingrel (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925-16930), distinct Na,K-A TPase kinetics are observed. Thus, at 1 mu M ATP, the effects of K+ on the Na-ATPase activity of alpha 2 and (alpha 1M32 are similar; both are activated, whereas alpha 1 and (alpha 3 are inhibited by the addit ion of K+ at low (0.1 mM) concentration. These effects are attributed to different rates of a step involved in K+ deocclusion (E(2)(K) <---- > E(1)K <---->, E(1) + K+) and are consistent with our earlier evidenc e (Wierzbicki, W., and Blostein, R. (1993) Proc. Natl. Acad. Sci. U. S . A. 90, 70-74) for a role of the NH2 terminus in the K+ deocclusion p athway of the Na,K-ATPase reaction. These differences are not directly related to differences in apparent affinities for ATP, since alpha 3 has alpha 1-like high affinity K+ inhibition but resembles (alpha 2 and (alpha 1M32 with respect to a lower K'(ATP).Na-ATPase activities o f alpha 2, alpha 3*, and alpha 1M32, but not alpha 1, are activated b y Li+ but not Rb+, consistent with a relatively faster rate of Li+ deo clusion (Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540), as well as higher affinity of alpha 3 for extracellu lar K+(Li+) activation of dephosphorylation (E(2)P + K+ <----> E(2)(K) + P-i). Inhibition of Na ATPase by higher concentrations (greater tha n or equal to mM) of K+ is observed with all isoforms and is attribute d to K+ acting at inhibitory cytoplasmic sites. Taken together, these results suggest that the weakening of intramolecular associations betw een the lysine-rich NH2 terminus and other parts of the protein, eithe r by removal of the terminus as in alpha 1M32 or by alteration in seco ndary structure as expected of the Gly-14-Gly-16 triplet in rat (alpha 2, alters the rate of the K+ deocclusion pathway of the Na,K-ATPase reaction mechanism.