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 Vmax 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+ K1/2 values for the activation of the Na,K-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 Ser775 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 Ser775 are required for efficient enzyme function. Moreover, these results suggest Ser775 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.