Specific protonation of acidic residues confers K⁺ selectivity to the gastric proton pump

The gastric proton pump (H⁺,K⁺-ATPase) transports a proton into the stomach lumen for every K⁺ ion exchanged in the opposite direction. In the lumen-facing state of the pump (E2), the pump selectively binds K⁺ despite the presence of a 10-fold higher concentration of Na⁺. The molecular basis for the ion selectivity of the pump is unknown. Using molecular dynamics simulations, free energy calculations, and Na⁺ and K⁺-dependent ATPase activity assays, we demonstrate that the K⁺ selectivity of the pump depends upon the simultaneous protonation of the acidic residues E343 and E795 in the ion-binding site. We also show that when E936 is protonated, the pump becomes Na⁺ sensitive. The protonation-mimetic mutant E936Q exhibits weak Na⁺-activated ATPase activity. A 2.5-Å resolution cryo-EM structure of the E936Q mutant in the K⁺-occluded E2-Pi form shows, however, no significant structural difference compared with wildtype except less-than-ideal coordination of K⁺ in the mutant. The selectivity toward a specific ion correlates with a more rigid and less fluctuating ion-binding site. Despite being exposed to a pH of 1, the fundamental principle driving the K⁺ ion selectivity of H⁺,K⁺-ATPase is similar to that of Na⁺,K⁺-ATPase: the ionization states of the acidic residues in the ion-binding sites determine ion selectivity. Unlike the Na⁺,K⁺-ATPase, however, protonation of an ion-binding glutamate residue (E936) confers Na⁺ sensitivity.