The Gastric Acid Pump as a Target for Ulcer Treatment

描述(申请人提供)：胃H+，K+ATPase催化胃酸分泌的最后一步，从而在小管膜上产生超过一百万倍的质子梯度。30年来，它一直是这个实验室的研究重点。近年来，我们感兴趣的是胃酸泵的结构-功能关系，以及共价抑制剂(质子泵抑制剂，PPI)和K+竞争性可逆抑制剂(酸泵拮抗剂，APAs)对该泵的抑制机制。在拟议的研究中，我们计划继续我们的定点突变方法，并以SERCA Ca ATPase的4个可用晶体结构为模板进行详细的同源性分析和建模，以更好地确定胃酸泵的转运。Ca-ATPase虽然只有29%与H+-K+-ATPase同源，但具有非常相似的整体结构，并使用膜结构域中的羧酸簇作为离子转运结合和进出口部位，与H+，K+ATPase有75%同源性的Na，K+ATP酶也是如此。我们计划通过分析各种酶的活性，包括所选择的突变位点的磷酸化和去磷酸化，来描述离子从细胞质到管腔的运输途径，以及K+从管腔到细胞质的运输途径。我们提出了一个假设，即唯一的Lys791插入到一个酸簇(D814，E820，E795)允许在所需的pH~1.0下释放质子，K+与腔内羰基结合取代了与Lys 791结合的两个羧酸，从而允许Iys 791返回并在这个位置被K+取代。新的突变体将通过同源模型进一步确定H+，K+ATPase的离子转运途径。由于酸泵拮抗剂正处于最终的临床试验中，我们计划通过合成一类新的化合物并确定其突变会改变这些新化合物的亲和力或抑制性质的氨基酸残基，来更准确地定义它们与酶的结合部位，就像我们对现在经典的咪唑-1，2a吡啶类化合物所做的那样，这些化合物经常显示出意想不到的负面副作用。由于酸性分泌调节的一个重要步骤涉及壁细胞的形态变化，其中ATPase从细胞质膜位置移动到分泌小管的微绒毛，我们将继续研究该酶稳定表达的亚单位在极化胃细胞中的运输和分类。此外，我们还将研究YFP-β亚单位敲入结构在小鼠胃、活小鼠胃腺和其他组织中的分布，如表达该酶但功能未知的肾脏。与(3亚基)相互作用的支架蛋白将使用分裂泛素方法来阐明，该方法能够定义与特定膜插入蛋白相关的那些蛋白。这些研究将有助于阐明泵的易位在调节胃酸分泌中的作用。这项拟议的研究结果将进一步提高用于治疗酸相关疾病的药物，以及我们对ATPase和参与其活性调节的细胞事件的了解。

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.