Mutations of Ser-23 of the alpha1 subunit of the rat Na+/K+-ATPase to negatively charged amino acid residues mimic the functional effect of PKC-mediated phosphorylation.

The Na+/K+-ATPase is a target protein for protein kinase C (PKC). The PKC-mediated phosphorylation of the rat alpha1 subunit at Ser-23 results in the inhibition of its transport function. To understand the molecular basis of the inhibition by PKC, the Ser-23 in the rat alpha1 subunit has been replaced by negatively (Asp, Glu) or positively (Lys) charged, or uncharged (Gln, Ala) residues, and the mutants were expressed in Xenopus oocytes. Ouabain-specific 86Rb uptake and pump-generated current as well as sensitivity to ouabain and to external K+ have been investigated. When Ser-23 was replaced by the negatively charged residues, transport function was inhibited, and simultaneously synthesis of the alpha subunits was enhanced. In addition, if Ser-23 was substituted by Glu, the K(I) value for inhibition of transport by ouabain was drastically increased from 46.5 microM to 1.05 mM. The data suggest that insertion of a negative charge within the N-terminus of alpha subunit of the Na+/K+-ATPase due to phosphorylation of Ser-23 plays an important role in the PKC-mediated inhibition of transport function.