Comparison of the Effects of Potassium on Ouabain Binding to Native and Site-Directed Mutants of Na,K-ATPase

We examined the effect of K+ on Mg2+- and Pi-supported [3H] ouabain binding to Na,K-ATPases, including partially purified enzyme from sheep kidney and wild-type and mutant sheep α1 isoforms (C104A, Y108A, E116Q, P118K, Y124F, R880P, R880L, and N122D) expressed in NIH3T3 cells. In the presence of increasing concentrations of K+, [3H] ouabain binding to these enzymes decreases but never reaches nonspecific binding levels, consistent with the concept that ouabain is still able to bind to the K+-complexed enzyme but with reduced affinity. A partially competitive model for K+ inhibition of ouabain binding is proposed which satisfactorily fits the binding data. The model is consistent with the sequential binding of two K+ ions to the enzyme. Ki values (≍ 1.0 mM) for K+ obtained from this model are comparable to the apparent K+ affinities of the rat α isoforms determined by measuring the K+ dependence of Na,K-ATPase activity [E. A. Jewell and J. B. Lingrel (1991) J. Biol. Chem. 266, 16925-16930]. This is consistent with the concept that K+ inhibition of Mg2+ plus Pi supported ouabain binding is mediated by K+ binding to the same high-affinity binding sites present in the native enzyme under physiological conditions. While the mutants exhibit binding constants for ouabain which vary more than 30-fold from that of the wild-type enzyme, their affinities for K+ differ less than twofold from that of the native enzyme. Our results suggest that the ouabain and K+ binding sites are not the same and are differentially affected by mutations of the enzyme. The system described here should prove useful in the analysis of cation binding to other mutants of the Na,K-ATPase, especially those carrying amino acid replacements which result in an inactive enzyme.