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 P-i-supported [H-3] ouabai n binding to Na,K-ATPases, including partially purified enzyme from sh eep kidney and wild-type and mutant sheep alpha 1 isoforms (C104A, Y10 8A, E116Q, P118K, Y124F, R880P, R880L, and N122D) expressed in NIH3T3 cells. In the presence of increasing concentrations of K+, [H-3] ouaba in binding to these enzymes decreases but never reaches nonspecific bi nding levels, consistent with the concept that ouabain is still able t o bind to the K+-complexed enzyme but with reduced affinity. A partial ly competitive model for K+ inhibition of ouabain binding is proposed which satisfactorily fits the binding data. The model is consistent wi th the sequential binding of two K+ ions to the enzyme. K-i values (ap proximate to 1.0 mM) for K+ obtained from this model are comparable to the apparent K+ affinities of the rat alpha isoforms determined by me asuring the K+ dependence of Na,K-ATPase activity [E. A. Jewell and J. B. Lingrel (1991) J. Biol. Chem. 266, 16925-16930]. This is consisten t with the concept that K+ inhibition of Mg2+ plus P-i supported ouaba in binding is mediated by K+ binding to the same high-affinity binding sites present in the native enzyme under physiological conditions. Wh ile 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 ar e differentially affected by mutations of the enzyme. The system descr ibed here should prove useful in the analysis of cation binding to oth er mutants of the Na,K-ATPase, especially those carrying amino acid re placements which result in an inactive enzyme. (C) 1995 Academic Press , Inc.