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

New homology models of the gastric H,K ATPase in the E 1 K and E 2 P states are presented as the first structures of a K + countertransport P 2 type ATPase identifying ion entry and exit paths. The new E 2 P 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 E 2 K. Concentrations of K + ~ten fold higher than the K m for ATPase activation inhibit the H,K ATPase. In contrast to the high affinity K + site for ATPase activation accessed from the extracytoplasmic surface (K m,app = 0.2 mM), this low affinity inhibitory site (K m,app = 8 mM) is accessed from the cytoplasmic surface. In order to explain K + exit to the cytoplasm from the E 1 K conformation of the H,K ATPase, a homology model was constructed based on the backbone coordinates of pdb.1su4 , the srCa ATPase in the E 1 2Ca 2+ conformation. Hydronium and K + were substituted for Ca 2+ 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 E 1 K conformation but not in E 2 K. VAGLHS and NIH DK 58333