The Cavity Structure for Docking the K+-competitive Inhibitors in the Gastric Proton Pump

Asano, Shinji; Yoshida, Ayumi; Yashiro, Hiroaki; Kobayashi, Yusuke; Morisato, Anna; Ogawa, Hirotaka; Takeguchi, Noriaki; Morii, Magotoshi

doi:10.1074/jbc.m308934200

Cited by 42 publications

(28 citation statements)

References 48 publications

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“…Mutations towards the exoplasmic surface of TM4, TM5 [3], TM6, the loop between TM5 and TM6, and one site at the end of TM8 altered either the K i or changed the nature of inhibition from strictly competitive to mixed or even non-competitive without affecting ion affinity. Mutation of lysine 791 to serine greatly reduced enzyme activity as well as increased the K i for SCH28080 [31,64,66].…”

Section: Acid Pump Antagonistsmentioning

confidence: 99%

The gastric HK-ATPase: structure, function, and inhibition

Shin

Munson

Vagin

et al. 2008

Pflugers Arch - Eur J Physiol

219

181

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The gastric H,K-ATPase, a member of the P 2 -type ATPase family, is the integral membrane protein responsible for gastric acid secretion. It is an α,β-heterodimeric enzyme that exchanges cytoplasmic hydronium with extracellular potassium. The catalytic α subunit has ten transmembrane segments with a cluster of intramembranal carboxylic amino acids located in the middle of the transmembrane segments TM4, TM5,TM6, and TM8. Comparison to the known structure of the SERCA pump, mutagenesis, and molecular modeling has identified these as constituents of the ion binding domain. The β subunit has one transmembrane segment with N terminus in cytoplasmic region. The extracellular domain of the β subunit contains six or seven Nlinked glycosylation sites. N-glycosylation is important for the enzyme assembly, maturation, and sorting. The enzyme pumps acid by a series of conformational changes from an E 1 (ion site in) to an E 2 (ion site out) configuration following binding of MgATP and phosphorylation. Several experimental observations support the hypothesis that expulsion of the proton at 160 mM (pH 0.8) results from movement of lysine 791 into the ion binding site in the E 2 P configuration. Potassium access from the lumen depends on activation of a K and Cl conductance via a KCNQ1/KCNE2 complex and Clic6. K movement through the luminal channel in E 2 P is proposed to displace the lysine along with dephosphorylation to return the enzyme to the E 1 configuration. This enzyme is inhibited by the unique proton pump inhibitor class of drug, allowing therapy of acid-related diseases.

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Section: Acid Pump Antagonistsmentioning

confidence: 99%

The gastric HK-ATPase: structure, function, and inhibition

Shin

Munson

Vagin

et al. 2008

Pflugers Arch - Eur J Physiol

219

181

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“…This would account for photoaffinity labeling of the M1M2 membrane pair by a p-azido derivative of SCH28080 (32). The interaction of Y799 with the inhibitor was investigated in recent site-specific mutagenesis studies identifying this residue as an important binding determinant (55). The binding site orientation of SCH28080 proposed by Asano et al (55), however, is rotated ~90°w ith respect to that shown in Figure 4C to make the imidazopyridine ring roughly coplanar with respect to the membrane plane.…”

Section: Inhibitor Binding To the New E 2 P Modelmentioning

confidence: 99%

“…The interaction of Y799 with the inhibitor was investigated in recent site-specific mutagenesis studies identifying this residue as an important binding determinant (55). The binding site orientation of SCH28080 proposed by Asano et al (55), however, is rotated ~90°w ith respect to that shown in Figure 4C to make the imidazopyridine ring roughly coplanar with respect to the membrane plane. More importantly, this proposed binding mode does not account for the complete elimination of binding in the A335C mutant.…”

Section: Inhibitor Binding To the New E 2 P Modelmentioning

confidence: 99%

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

2007

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New 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 + counter-transport P 2 -type ATPase exhibiting ion entry and exit paths. Homology modeling was first used to generate a starting conformation from the srCa ATPase E 2 P form (PDB code 1wpg) that contains bound MgADP. Energy minimization of the model showed a conserved adenosine site but nonconserved polyphosphate contacts compared to the srCa ATPase. Molecular dynamics was then employed to expand the luminal entry sufficiently to allow access of the rigid K + competitive naphthyridine inhibitor, Byk99, to its binding site within the membrane domain. The new E 2 P model had increased 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. A K + exit path to the cytoplasm operating during the normal catalytic cycle is also proposed on the basis of an E 1 K homology model derived from the E 1 2Ca 2+ form of the srCa ATPase (PDB code 1su4). Autodock analyses of the new E 2 P model now correctly discriminate between high-and low-affinity K + competitive inhibitors. Finally, the expanded luminal vestibule of the E 2 P model explains high-affinity ouabain binding in a mutant of the H,K ATPase P 2 -type ATPases (ion pumps) catalyze active ion transport by coupling autophosphorylation and dephosphorylation to ion movement across lipid bilayers. The Na,K ATPases and the gastric H,K ATPase couple outward transport of sodium or protons to the inward transport of potassium. They are distinguished from the other members of this family by the presence of a glycosylated β subunit tightly bound to the larger catalytic (α) subunit and by the need for K + on their luminal surface for completion of the catalytic cycle. There is about 62% homology between their α subunits and about 35% homology between the gastric β subunit and the Na,K ATPase β 2 isoform (1). The α subunits contain the known binding sites for ATP, ions, and inhibitors. Several avenues of research have defined the biochemical features of the shared transport mechanism (2). Transport is achieved through a series of conformational transitions which alternatively bind the transported ions from the cytoplasm in the E 1 form or from the lumen after autophosphorylation from ATP to give the E 2 P conformer. Conversion to E 2 P is associated with export of the outbound cation with generation of luminal acid in the case of the H,K ATPase. For Na,K and H,K ATPases binding of potassium activates dephosphorylation to give a state with tightly bound ion (E 2 K occluded) in equilibrium with † Supported in part by the U.S. Veterans Administration and NIH Grants DK46917, DK53462, and DK58333. *Corresponding author. . kmunson@ucla.edu. ‡ David ...

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“…For numbering the amino acid sequence of the gastric H ϩ ,K ϩ -ATPase ␣-subunit, the cDNA sequence of the rabbit gastric H ϩ ,K ϩ -ATPase ␣-subunit (GenBank TM accession number P27112) was adopted. Site-directed mutagenesis was performed using the QuikChange II site-directed mutagenesis kit as described previously (21). The mutated cDNA sequences were verified using a Long-Read Tower DNA sequencer (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 Charge Transfer Pathway in Proton Transport of Gastric H+,K+-ATPase

Morii

Yamauchi

Ichikawa

et al. 2008

Journal of Biological Chemistry

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Gastric؉ ,K ؉ -ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H 3 O ؉ -Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H 2 O in sites 2 and 1, the H 2 O comes from cytosolic medium, and H 3 O ؉ in the sites are transported into lumen during the conformational transition from E 1 P to E 2 P.

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The Cavity Structure for Docking the K+-competitive Inhibitors in the Gastric Proton Pump

Cited by 42 publications

References 48 publications

The gastric HK-ATPase: structure, function, and inhibition

The gastric HK-ATPase: structure, function, and inhibition

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

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 Charge Transfer Pathway in Proton Transport of Gastric H+,K+-ATPase

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