Nonpolar amino acid substitutions of potential cation binding residues glu-955 and glu-956 of the rat alpha 1 isoform of Na+, K(+)-ATPase.

Side chain oxygens are critical in the binding of cations by several macromolecules, and chemical labeling studies suggest that the carboxyl oxygens of E953 of the pig kidney Na, K-ATPase are essential for Na+ and K+ ligation. An adjacent residue, E954, is highly conserved and may also be important in cation binding. Replacement of the corresponding glutamates in the rat alpha 1 isoform (E955 and E956) with glutamines or aspartic acids has only a modest effect on enzyme activity, but both substituted amino acids contain at least one side chain oxygen, and may fulfill the role of the wild-type glutamates in binding cations. Since substitutions of amino acids lacking an oxygenated side chain have not been made at positions 955 and 956, nonpolar amino acid replacements (E955A, E956A, E955A-E956A, and E955L-E956L) were made, and the effects of the substitutions on the cation dependence properties of the mutant enzymes were examined. The substitutions were made using site-directed mutagenesis of the rat alpha 1 cDNA (which encodes a ouabain-resistant alpha subunit), followed by transfection of wild-type and mutant cDNAs into ouabain-sensitive HeLa cells, with subsequent expression of the altered proteins. Transfection with all cDNA constructs resulted in the ouabain resistance of transfected HeLa cells, demonstrating that the modified Na, K-ATPase in each case was functional. Ouabain resistance of Na, K-ATPase activity was increased 1,000-fold in microsomal membranes isolated from cells transfected with wild-type rat alpha 1 (WT) and all mutant cDNA constructs, compared to activity in membranes prepared from untransfected HeLa cells. This confirmed the expression of a ouabain-resistant enzyme in all transfected cell lines, and the enhanced ouabain resistance permitted the study of the exogenous, expressed Na, K-ATPase in the presence of 5.0 microM ouabain. Cation stimulation of exogenous Na, K-ATPase activity was not affected by the E955A substitution and only slightly by the E956A replacement (K0.5(Na+) of 3.40 +/- 0.21, 3.30 +/- 0.22, and 6.60 +/- 0.55 mM NaCl for WT, E955A and E956A, respectively; K0.5(K+) of 0.78 +/- 0.01, 0.74 +/- 0.10, and 0.56 +/- 0.11 mM KCl). Even doubly substituted enzymes had only mild alterations in cation dependence properties (K0.5(Na+) of 7.54 +/- 0.23, 7.14 +/- 0.10 mM NaCl for E955A-E956A, E955L-E956L, respectively; K0.5(K+) of 0.43 +/- 0.13, 1.83 +/- 0.13 mM KCl). The results demonstrate that there are no requirements for an oxygenated side chain at positions 955 and 956 for normal or nearly normal cation dependence.