Study on the Structure and Mechanism Involved in Functional regulation of Gastric Proton Pump

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The gastric H^+,K^<+->ATPase is the proton pump responsible fbr gastric acid secretion. This pump consists of the catalytic α-and non-catalytic β-subunits. In this research project, we constructed stable cell lines expressing the gastric proton pump, and studied the finctional regulation mechanisms by site-directed mutagenesis in combination with homology modeling method.(1) We constructed three kinds of stable cell lines; the a-expressing cells, the [3-expressing cells, and the α+β-expressing cells. The α-subunit was retained in the intracellular compartment, and no cell surface expression was observed in the absence of the β-subunit. On the other hand, cell surface expression of the β-subunit was observed even in the absence of the α-subunit. Cell surface expression of the α-and β-subunits was observed in the α+β-expressing cells. The α+β-expressing cells represented rubidium (^<86>Rb) and proton transport activities, which were inhibited by inhibitors of prothn pump.(2) We studied t … More he binding site of acid pump antagonist (reversible inhibitor of the gastric proton pump), SCH 28080 by site-directed mutagenesis in combination with homology modeling method. When Tyr-801 on the M5 transmembrane segment of the a-subunit was replaced by alanine, the mutant (Y801A) showed 60 times lower sensitivity to SCH 28080 compared with that of wild type. The sensitivity to SCH 28080 was dependent on the bulkiness of this residue, indicating that the side chain of this residue is important for the interaction with this inhibitor. In the 3-D models of the E_2 and E_2P conformations of α-subunit, Tyr-801 is located at a top surface of a docking pocket of the luminal cavity, putative binding site of SCH 28080, surrounded by the TM1, TM4, TMS, TM6 and TM8 segments and the M5/M6, M7/M8 and M9/M10 luminal loops.(3) We studied the molecular mechanisms of the proton transfer by the gastric proton pump. Homology modeling and molecular dynamics calculation of the α-subunit indicate that protons are transferred from the basic amino acid residue in the cytoplasmic side to the acidic amino acid residue in the cation binding site via the polar amino acid residues. On the other hand, in the estimation of water transport activity in the hog GI gastric vesicles, we found that 1.8 mol of water were transported by 1 mol of ATP hydrolysis, and that this water transport was completely inhibited by an inhibitor ofprotonpump, SCH 28080. From these findings, we concluded that proton transport is carried out by charge movement between the polar amino acid residues in the cytoplamsic half, and that the oxonium ions (H_3O+) formed in the cation binding sites are transported in the luminal half of the membrane. Less