The Gastric Acid Pump as a Target for Ulcer Treatment

描述（由申请人提供）：胃H+， K+ atp酶催化胃酸分泌的最后一步，从而产生超过一百万倍的质子梯度穿过小管膜。这是这个实验室30年来的研究重点。近年来，我们的兴趣主要集中在该泵的结构-功能关系以及共价抑制剂（质子泵抑制剂，PPIs）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位点定向诱变方法，结合详细的同源性分析和建模，使用SERCA Ca atp酶的4种可用晶体结构作为模板，以更好地定义胃酸泵的转运。Ca atp酶虽然与H+，K+ - atp酶只有29%的同源性，但具有非常相似的整体结构，并且也使用膜结构域的羧酸簇作为离子运输结合和进出口位点，而Na， K+ atp酶与H+，K+ atp酶同源性为75%。我们计划通过分析各种酶的活性，包括磷酸化和去磷酸化，来描绘水合氢离子从细胞质转运到管腔和K+从管腔转运到细胞质的途径。我们提出了一个假设，即Lys791独特的插入到一个酸簇（D814, E820, E795）中，可以在所需的pH~1.0下释放质子，并且K+结合到腔内羰基上取代了与Lys791结合的两个羧酸，从而允许fiys791返回并在该位点被K+取代。新的突变体将通过同源性建模进一步定义H+，K+ atp酶的离子转运途径。由于酸泵拮抗剂处于最后的临床试验阶段，我们计划通过合成一类新的化合物和鉴定突变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精确地确定它们与酶的结合位点，就像我们对现在经典的咪唑-1,2a prydine类所做的那样，它们经常表现出意想不到的负面副作用。由于酸分泌调节的一个重要步骤涉及壁细胞的形态转化，其中atp酶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在极化胃细胞中稳定表达的亚基的运输和分选。此外，我们将研究YFP-¿亚基敲入构建体在小鼠胃、活体小鼠胃腺和其他组织（如肾脏）中的分布，其中酶表达但功能未知。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酸分泌中的作用。拟议的研究结果将进一步改善用于治疗酸相关疾病的药物，以及我们对atp酶和参与其活性调节的细胞事件的认识。

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.