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

• Published in: The Journal of biological chemistry Vol. 300; no. 1; p. 105542
• Main Authors: Madapally, Hridya Valia, Abe, Kazuhiro, Dubey, Vikas, Khandelia, Himanshu
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01.01.2024
• Subjects: free energy perturbation; H+,K+-ATPase; ion selectivity; proton pump; Cryo-EM; membrane protein; molecular dynamics
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2023.105542

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 to wild type except less than ideal coordination of K in the mutant. The selectivity towards 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 ionisation 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.