Modulation of the Na,K-ATPase by Magnesium Ions

Since the beginning of investigations of the Na,K-ATPase, it has been well-known that Mg²⁺ is an essential cofactor for activation of enzymatic ATP hydrolysis without being transported through the cell membrane. Moreover, experimental evidence has been collected through the years that shows that Mg²⁺ ions have a regulatory effect on ion transport by interacting with the cytoplasmic side of the ion pump. Our experiments allowed us to reveal the underlying mechanism. Mg²⁺ is able to bind to a site outside the membrane domain of the protein’s α subunit, close to the entrance of the access channel to the ion-binding sites, thus modifying the local concentration of the ions in the electrolyte, of which Na⁺, K⁺, and H⁺ are of physiological interest. The decrease in the concentration of these cations can be explained by electrostatic interaction and estimated by the Debye–Hückel theory. This effect provokes the observed apparent reduction of the binding affinity of the binding sites of the Na,K-ATPase in the presence of various Mg²⁺ concentrations. The presence of the bound Mg²⁺, however, does not affect the reaction kinetics of the transport function of the ion pump. Therefore, stopped-flow experiments could be performed to gain the first insight into the Na⁺ binding kinetics on the cytoplasmic side by Mg²⁺ concentration jump experiments