SUBSTITUTIONS OF SERINE-775 IN THE ALPHA-SUBUNIT OF THE NA,K-ATPASE SELECTIVELY DISRUPT K+ HIGH-AFFINITY ACTIVATION WITHOUT AFFECTING NA+ INTERACTION

The functional role of serine 775, predicted to be located in the fifth transmembrane segment of the alpha subunit of the Na,K-ATPase (YTLTSNIPE), was studied using site-directed mutagenesis, expression, and kinetic analysis. Substitutions S775A, S775C, and S775Y were introduced into an ouabain-resistant-alpha 1 sheep isoform and expressed in HeLa cells. cDNAs carrying substitutions S775C and S775A produced ouabain-resistant colonies only when extracellular K+ was increased from 5.4 mM to 10 or 20 mM, respectively. No ouabain-resistant colonies were obtained for substitutions S775Y at any tested K+ concentration. Kinetic characterization of S775C and S775A substituted enzymes showed expression levels higher than control enzyme, reduced V-max and turnover, and normal phosphorylation and high affinity ATP binding. Dephosphorylation experiments indicated that S775A substituted enzyme is insensitive to ADP but readily dephosphorylated by K+. The K+ K-1/2 values for the activation of the Na,R-ATPase were markedly altered, with S775C displaying a 13-fold increase and S775A exhibiting a 31-fold increase. These large changes in the Na,K-ATPase affinity for K+ are consistent with the participation of this amino acid in binding K+ during the translocation of this cation. Substitutions of Ser(775) did not change Na+ affinity, indicating that this residue is likely not involved in Na+ binding and occlusion. These data show that the electronegative oxygen and the small side chain of Ser(775) are required for efficient enzyme function. Moreover, these results suggest Ser(775) plays a distinct role in K+ transport and not in Na interactions, revealing a possible mechanism for the enzymatic differentiation of these cations by the Na, K-ATPase.