Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: I. Sodium Binding is Associated with a Conformational Rearrangement

Abstract.

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 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 E₁/E₂ 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.