Isoform-specific effects of charged residues at borders of the M1-M2 loop of the Na,K-ATPase alpha subunit

The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain . Via a largely undefined mechanism, the ouabain affinity of the Na,K-ATPas e can be manipulated by mutating the residues at the borders of the first e xtracellular (M1-M2) loop of the a subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue , we compared the effects of two combinations of charged residues at the M1 -M2 loop border, R113,D124 and D113,R124 (numbered according to the rat alp ha 1 subunit), on the ouabain sensitivity of the alpha 1 and alpha 2 isofor ms. We report that ouabain sensitivity is dependent not only upon the ident ity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chi meric alpha subunits reveals that the effects of potassium are determined p rimarily by the interaction of the N-terminus and M1-M2 loop with the C-ter minal third of the a subunit. M1-M2 loop border residues may, therefore, in fluence ouabain sensitivity indirectly by altering the stability or structu re of the intermediate of the Na,K-ATPase catalytic cycle which is competen t to bind ouabain.