Site-directed mutagenesis was used to examine the importance of five carboxyl-containing amino acids localized in the putative membrane-spanning regions of the Na,K-ATPase (i.e., E327, E778, D803, D807, and D925 of the rat a2 isoform). The substitutions were introduced into a cDNA encoding a ouabain-resistant isoform (i.e., rat alpha2* which was mutated to encode a ouabain-resistant isoform), and the effect of these substitutions on Na,K-ATPase function was assessed by screening the altered enzymes for their ability to confer ouabain resistance when expressed in otherwise ouabain-sensitive cells. The expression of the a isoform containing certain substitutions at positions 327 and 925 was able to confer ouabain resistance to HeLa cells while the expression of rat alpha2* containing substitutions at positions 778, 803, and 807 was not. In particular, amino acids in each of these positions were substituted with leucine to evaluate the importance of the carboxyl-containing side chain. The ability of rat alpha2* containing E327L and D925L to confer ouabain resistance to HeLa cells indicates that neither the negative charge nor the oxygen-containing side chain is absolutely essential for overall function in this position. In contrast, the inability of rat alpha2* carrying E778L, D803L, and D807L to confer ouabain resistance suggests that the naturally occurring amino acid may be more critical structurally and/or functionally for the Na,K-ATPase. Other more conservative substitutions introduced to further characterize the role of particular amino acid side chains include E327D, E327Q, D803N, D803E, and D925N. Substitutions E327Q and D925N allowed the enzyme to retain function, in agreement with the effects of the leucine substitutions, while E327D, D803E, and D803N caused inactivation of the enzyme. The observation that Na,K-ATPase retains function when E327 is substituted with leucine and glutamine, but not aspartic acid, suggests that the size or the nature of the side chain may be important in this position for overall function. Cation dependence of Na,K-ATPase activity was assessed in enzyme containing substitutions which retained function. These results reveal that the Na+ and K+ stimulation of Na,K-ATPase activity is clearly different for rat alpha2* than for rat alpha2* containing the substitutions E327L, E327Q, D925L, and D925N. This study represents the first report of the effects of amino acid substitutions to five carboxyl-containing transmembrane residues in the Na,K-ATPase.
