Analysis of the Gastric H,K ATPase for Ion pathways and Inhibitor Binding Sites

Abstract

New homology models of the gastric H,K ATPase in the E1K and E2P states are presented as the first structures of a K+ countertransport P2 type ATPase identifying ion entry and exit paths. The new E2P model showed separation between transmembrane segments M3 through M8 and addition of water in this space showed not only an inhibitor entry path to the luminal vestibule but also a channel leading to the ion binding site. Addition of K+ to the hydrated channel with molecular dynamics modeling of ion movement identified a pathway for K+ from the lumen to the ion binding site to give E2K. Concentrations of K+ ~ten fold higher than the Km for ATPase activation inhibit the H,K ATPase. In contrast to the high affinity K+ site for ATPase activation accessed from the extracytoplasmic surface (Km,app = 0.2 mM), this low affinity inhibitory site (Km,app = 8 mM) is accessed from the cytoplasmic surface. In order to explain K+ exit to the cytoplasm from the E1K conformation of the H,K ATPase, a homology model was constructed based on the backbone coordinates of pdb.1su4, the srCa ATPase in the E12Ca2+ conformation. Hydronium and K+ were substituted for Ca2+ in sites I and II. The possibility that mutants of Q159 and E160 would affect activity and apparent ion affinity in a manner consistent with a role in the ion exit mechanism was investigated. These conserved residues are in close proximity to the ion gating residue, E343, in the E1K conformation but not in E2K. VAGLHS and NIH DK 58333