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. ? ?
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