Ion selectivity of the cytoplasmic binding sites of the Na,K-ATPase: I. Sodium binding is associated with a conformational rearrangement

To investigate Na+ binding to the ion-binding sites presented on the cytoplasmic side of the Na,K-ATPase, equilibrium Na+-titration experiments were performed using two fluorescent dyes, RH421(1) and FITC, to detect protein-specific actions. Fluorescence changes upon addition of Na+ in the presence of various Mg2+ concentrations were similar and could be fitted with a Hill function. The half-saturating concentrations and Hill coefficients determined were almost identical. As RH421 responds to binding of a Na+ ion to the third neutral site whereas FITC monitors conformational changes in the ATP-binding site or its environment, this result implies that electrogenic binding of the third Na+ ion is the trigger for a structural rearrangement of the ATP-binding moiety. This enables enzyme phosphorylation, which is accompanied by a fast occlusion of the Na+ ions and followed by the conformational transition E-1/E-2 of the protein. The coordinated action both at the ion and the nucleotide binding sites allows for the first time a detailed formulation of the mechanism of enzyme phosphorylation that occurs only when three Na+ ions are bound.