K+ Congeners That Do Not Compromise Na+ Activation of the Na+,K+-ATPase

The Na+,K+-ATPase is essential for ionic homeostasis in animal cells. The dephosphoenzyme contains Na+ selective inward facing sites, whereas the phosphoenzyme contains K+ selective outward facing sites. Under normal physiological conditions, K+ inhibits cytoplasmic Na+ activation of the enzyme. Acetamidinium (Acet+) and formamidinium (Form+) have been shown to permeate the pump through the outward facing sites. Here, we show that these cations, unlike K+, are unable to enter the inward facing sites in the dephosphorylated enzyme. Consistently, the organic cations exhibited little to no antagonism to cytoplasmic Na+ activation. Na+,K+-ATPase structures revealed a previously undescribed rotamer transition of the hydroxymethyl side chain of the absolutely conserved Thr772 of the α-subunit. The side chain contributes its hydroxyl to Na+ in site I in the E1 form and rotates to contribute its methyl group toward K+ in the E2 form. Molecular dynamics simulations to the E1·AlF4−·ADP·3Na+ structure indicated that 1) bound organic cations differentially distorted the ion binding sites, 2) the hydroxymethyl of Thr772 rotates to stabilize bound Form+ through water molecules, and 3) the rotamer transition is mediated by water traffic into the ion binding cavity. Accordingly, dehydration induced by osmotic stress enhanced the interaction of the congeners with the outward facing sites and profoundly modified the organization of membrane domains of the α-subunit. These results assign a catalytic role for water in pump function, and shed light on a backbone-independent but a conformation-dependent switch between H-bond and dispersion contact as part of the catalytic mechanism of the Na+,K+-ATPase. Background: The Na+,K+-ATPase discriminates between similar and abundant ions. Results: The K+ congener acetamidinium interacts with the outward facing sites of Na+,K+-ATPase, but does not interact with the inward facing sites. Conclusion: Water in the ion binding cavity regulates ion selectivity of the Na+,K+-ATPase. Significance: This study identifies new determinants of ion selectivity of K+-transporting P-type pumps.