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

胃H^+，K^<+-> atp酶是负责胃酸分泌的质子泵。该泵由催化α亚基和非催化β亚基组成。本课题通过构建稳定的胃质子泵表达细胞系，结合同源性建模方法，通过定点诱变研究其功能调控机制。(1)构建了三种稳定的细胞系；a-表达细胞、[3]表达细胞和α+β表达细胞。α-亚基保留在细胞内腔室中，缺乏β-亚基时细胞表面无表达。另一方面，即使在α-亚基缺失的情况下，细胞表面也有β-亚基的表达。α+β-表达的细胞表面可见α和β-亚基的表达。α+β-表达细胞代表铷（^<86>Rb）和质子转运活性，这些活性被质子泵抑制剂抑制。(2)结合同源性建模方法，采用定点诱变的方法研究了酸泵拮抗剂（胃质子泵可逆抑制剂）SCH 28080的结合位点。当a亚基M5跨膜段上的tyr1 -801被丙氨酸取代时，突变体Y801A对SCH 28080的敏感性比野生型低60倍。对SCH 28080的敏感性取决于该残基的体积，这表明该残基的侧链对于与该抑制剂的相互作用很重要。在α-亚基E_2和E_2P构象的三维模型中，tyr1 -801位于腔腔对接袋的顶表面，被TM1、TM4、TMS、TM6和TM8节段以及M5/M6、M7/M8和M9/M10腔袢所包围，推测为SCH 28080的结合位点。(3)研究了胃质子泵质子转移的分子机制。α-亚基的同源性建模和分子动力学计算表明，质子通过极性氨基酸残基从细胞质侧的碱性氨基酸残基转移到阳离子结合位点的酸性氨基酸残基。另一方面，在猪GI胃囊泡的水转运活性估计中，我们发现1 mol ATP水解可转运1.8 mol水，而这种水转运被质子泵抑制剂SCH 28080完全抑制。根据这些发现，我们得出结论，质子的运输是通过细胞质中一半极性氨基酸残基之间的电荷运动进行的，而在阳离子结合位点形成的氧离子（H_3O+）在膜的腔内部分运输。少

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