The Gastric Acid Pump as a Target for Ulcer Treatment

胃酸泵作为溃疡治疗的目标

• 批准号: 7484993
• 负责人: George Sachs
• 金额: $ 23.65万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7484993
• 关键词: ATP phosphohydrolase; Acids; Adverse effects; Affinity; Amino Acids; Binding; Binding Sites; Carboxylic Acids; Cell membrane; Cells; Class; Clinical Trials; Coupled; Cytoplasm; Disease; Enzymes; Event; Gastric Acid; Gastric Glands; Gastric Parietal Cells; H(+)-K(+)-Exchanging ATPase; Homology Modeling; Ion Transport; Kidney; Knock-in Mouse; Knowledge; Laboratories; Life; Location; Membrane; Methods; Modeling; Mus; Mutation; Na(+)-K(+)-Exchanging ATPase; Nature; Pathway interactions; Phosphorylation; Protein Dephosphorylation; Proteins; Proton Pump Inhibitors; Protons; Pump; Regulation; Research; Role; Scaffolding Protein; Site; Site-Directed Mutagenesis; Sorting - Cell Movement; Stomach; Structure; Structure-Activity Relationship; Ubiquitin; Ulcer; carbonyl group; cellular microvillus; enzyme activity; gastrointestinal microvillus; hydronium ion; improved; inhibitor/antagonist; interest; mutant; trafficking

DESCRIPTION (provided by applicant): The gastric H+, K+ ATPase catalyzes the final step of gastric acid secretion thereby generating a proton gradient across the canalicular membrane of greater than a million fold. It has been a focus of this laboratory for 30 years. Our interest in recent years has been the structure-function relationships of this pump and the mechanism of inhibition of this pump by covalent inhibitors (the proton pump inhibitors, PPIs) and the K+ -competitive reversible inhibitors (the acid pump antagonists, APAs).In the proposed studies, we plan to continue our site-directed mutagenesis approach coupled with detailed homology analysis and modeling using the 4 available crystal structures of the SERCA Ca ATPase as a template to better define transport by the gastric acid pump. The Ca ATPase although only 29% homologous to the H+-K+-ATPase, has a very similar overall structure and also uses carboxylic acid clusters in the membrane domain as the ion transport- binding and export-import sites as does the Na, K+ ATPase that is 75% homologous to the H+,K+ ATPase. We plan to delineate pathways for transport of hydronium ion from cytoplasm to lumen and K+ from lumen to cytoplasm by analyzing various enzyme activities including phosphorylation and dephosphorylation of selected site mutants. We have developed a hypothesis that the unique Lys791 insertion into one cluster of acids (D814, E820, E795) allows release of proton at the required pH~1.0 and K+ binding to luminal carbonyl groups displaces two of the carboxylic acids bound to lys 791, thereby allowing return ofIys791 and replacement by K+ at this site. New mutants will further define the ion transport pathways of the H+,K+ ATPase by homology modeling. Since acid- pump antagonists are in final clinical trials, we plan to define their site of binding to the enzyme more precisely by synthesizing a new class of compounds and identifying the amino acid residues whose mutation alters the affinity or nature of inhibition by these new compounds as we have done for the now classical imidazo-1,2a prydine class that often show unexpected negative side effects. Since an important step of acid secretory regulation involves a morphological transformation of the parietal cell wherein the ATPase moves from a cytoplasmic membrane location to the microvilli of the secretory canaliculus, we will continue our study of trafficking and sorting of the stably expressed ¿ subunit of the enzyme in polarized gastric cells. In addition, we will study the distribution of a YFP- ¿ subunit knock in- construct in the mouse stomach, living mouse gastric glands and in other tissuess such as the kidney where the enzyme is expressed but function is unknown. The scaffold proteins interacting with the (3 subunit will be elucidated using the the split ubiquitin method which is capable of defining those proteins associated with a particular membrane inserted protein. These studies will aid in clarifying the role of translocation of the pump in regulation of acid secretion. The results of the proposed research will further improve the agents used for the treatment of acid-related diseases and also our knowledge of the ATPase and cellular events involved in regulation of its activity.

描述（由适用提供）：胃H+，K+ ATPase催化胃酸分泌的最后一步，从而在大于一百万倍的大口腔膜上产生质子梯度。这是该实验室的重点已有30年了。近年来，我们的兴趣是该泵的结构功能关系以及共价抑制剂（Proton泵抑制剂，PPI）和K+竞争性可逆抑制剂（酸泵拮抗剂，APA，APA）的结构功能机制。在建议的研究中，我们计划使用详细的分析型号，我们计划进行详细培训，我们计划进行详细培训，我们计划进行详细的分析。 SERCA CA ATPase的结构作为模板，以更好地定义胃酸泵的运输。 The Ca ATPase is only 29% homologous to the H+-K+-ATPase, has a very similar overall structure and We have developed a hypothesis that the unique Lys791 insertion into one cluster of acids (D814, E820, E795) allows release of proton at the required pH~1.0 and K+ binding to luminal carbonyl groups displaces two of the carboxylic acids bound to lys 791, thereby允许在此站点返回iys791并替换K+。新突变体将通过同源性建模进一步定义H+，K+ ATPase的离子传输途径。由于酸 - 泵拮抗剂是在最终临床试验中，因此我们计划通过合成一类新化合物并鉴定氨基酸保留其突变会改变这些新化合物的亲和力或抑制性质的性质，从而更精确地定义其与酶结合的位点，因为我们对我们现在对现在的经典imidazo-1,2a pry sand sande canse diffest canse diffest canse diffest Sears table sance diffest cance diffest cance。由于酸性分泌调节的重要步骤涉及对顶酶的形态转化，其中ATPase从细胞质膜的位置移动到秘密的小管的微壁，我们将继续研究稳定表达的eNzyme在极化气体中的enzyme的贩运和分类。此外，我们将研究小鼠摊位中的YFP-亚基敲击构建体的分布，活的小鼠胃腺以及在表达酶的肾脏等其他组织中的分布，但功能尚不清楚。 The scaffold proteins interacting with the (3 subunits will be elucidated using the split ubiquitin method which is capable of defining those proteins associated with a particular membrane inserted protein. These studies will aid in clariifying the role of translocation of the pump in regulation of acid secretion. results of the proposed research will further improve the agents used for the treatment of acid-related diseases and also our knowledge of the ATPase and cellular events involved in调节其活性。