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

胃H^+，K^&lt；+-&Gt；ATPase是负责FBR胃酸分泌的质子泵。该泵由催化的α亚基和非催化的β亚基组成。在本研究中，我们构建了稳定表达胃质子泵的细胞系，并结合同源模建的方法，利用定点突变的方法对其功能调控机制进行了研究。(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，在没有β亚基的情况下没有观察到细胞表面的表达。另一方面，即使在没有β亚基的情况下，也能观察到α亚基在细胞表面的表达。在表达α的细胞中可观察到β-和α+β-亚基的细胞表面表达。表达α+β的细胞代表Rb(^&lt；86&gt；Rb)和质子转运活性，这两种活性被质子泵抑制剂抑制。(2)我们研究了t…通过定点突变和同源模建相结合的方法，获得了更多的酸性泵拮抗剂(胃质子泵的可逆抑制物)的结合位点，SCH 28080。用丙氨酸取代α-亚基M5跨膜片段上的酪氨酸后，突变株(Y801A)对SCH 28080的敏感性比野生型降低60倍。对SCH 28080的敏感性取决于该残基的体积，表明该残基的侧链对与该缓蚀剂的相互作用是重要的。在α亚基的E_2和E_2P构象的三维模型中，TYR-801位于腔腔对接口袋的顶面，推测是SCH 28080的结合部位，被TM1、TM4、TMS、TM6和TM8片段以及M5/M6、M7/M8和M9/M10腔环包围。(3)研究了胃质子泵质子转移的分子机制。α亚基的同源模建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一侧的碱性氨基酸残基转移到阳离子结合部位的酸性氨基酸残基。另一方面，在估计猪胃小泡的水转运活性时，我们发现1.8摩尔的水被1摩尔的三磷酸腺苷水解所转运，并且这种水的转运完全被质子泵抑制剂SCH 28080所抑制。根据这些发现，我们得出结论，质子的运输是通过细胞质一半中极性氨基酸残基之间的电荷运动进行的，而在阳离子结合部位形成的氧离子(H_3O+)是在膜的管腔半部分中运输的。较少

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