<p>The Na⁺,K⁺-ATPase is an integral membrane protein present in all animal plasma membranes. It uses the energy of ATP hydrolysis to pump 3 Na⁺ ions per ATP hydrolysed from the cell cytoplasm into the extracellular fluid in exchange for 2&#xa0;K⁺ ions. The Na⁺ electrochemical potential gradient it produces is used as an energy source to drive all animal secondary transporters, for example, in nutrient reabsorption in the kidney. In this synthesis of new and previously published results, it is shown that the rate of the mammalian enzyme’s rate-determining E2 → E1 conformational change, during which K⁺ ions are released to the cytoplasm, is strongly dependent on an electrostatic interaction in the E2 state. This electrostatic interaction must be broken to allow the enzyme to convert to the E1 state and allow Na⁺ pumping. The strength of the interaction depends on ionic strength and, even more strongly, on the concentration of divalent metal ions, i.e., Ca²⁺ or Mg²⁺. A likely candidate for the interaction is between the protein’s positively-charged lysine-rich N-terminus and the negatively-charged membrane cytoplasmic surface. Comparison of the Ca²⁺ and Mg²⁺ dissociation constants measured with the physiological levels of the two ions suggests that Mg²⁺, but not Ca²⁺, could play a regulatory role for the Na⁺,K⁺-ATPase.</p> Graphical Abstract <p></p>