Structure and function of novel pumps and channels in gastrointestinal tract

1.我们成功构建了一个稳定表达胃质子泵α和β亚基的细胞系（极化LLC-PK1细胞）。利用该细胞系，我们首次发现胃质子泵具有磷脂翻转酶的功能。在对表达胃质子泵的HEK-293细胞的研究中，我们发现在M6片段中对质子泵功能重要的氨基酸残基完全定位在假定的α-螺旋结构的一侧。我们还发现，β-亚基外域的所有三个二硫键对于α-β的组装、质子泵活性、α和β-亚基的稳定性以及α-亚基的细胞表面递送都是必需的。我们成功地建立了条件永生化的胃粘膜细胞系，表达胃质子泵mRNA，该细胞系是由含有温度敏感的猴病毒40大t抗原的转基因小鼠制备的。我们发现IL-1β与兔胃壁细胞的IL-1受体结合，并通过G蛋白介导的Rho/Rho激酶依赖的O_2^-的产生抑制基底外侧家政Cl^-通道。CLCA1不是胃内务Cl^-通道的亚基。我们发现了一氧化氮依赖性大鼠结肠粘膜Cl^-通道激活的新机制。在这个机制中，NO刺激血栓素A_2的产生，释放的血栓素A_2激活结肠隐窝细胞的Cl^-通道。

1.We succeeded to construct a stable cell line(polarized LLC-PK1 cell) expressing the gastric proton pump α-and β-subunits. By using this cell line, we found for the first time that the gastric proton pump functions as phospholipid flippase.2.In the studies using HEK-293 cells expressing gastric proton pump, we found that the amino acid residues important for the proton pump function in the M6 segment are exclusively localized on the one side of the putative α-helical structure. We also found that all three disulfide bonds in the ectodomain of the β-subunit are necessary for the α-β assembly, proton pump activity, stability of the α-and β-subunits, and cell surface delivery of the α-subunit.3.We succeeded to establish conditionally immortalized gastric mucosal cell lines expressing mRNA for gastric proton pump from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen.4.We found that IL-1β binds to the IL-1 receptor of rabbit gastric parietal cells and inhibits the basolateral housekeeping Cl^-channel via G protein-mediated Rho/Rho-kinase-dependent production of O_2^-. CLCA1 is not a subunit of the gastric housekeeping Cl^-channel.5.We found that the NO-dependent novel mechanism of activation of Cl^-channels in rat colonic mucosa. In this mechanism, NO stimulates the production of thromboxane A_2, and the released thromboxane A_2 activates the Cl^-channels in colonic crypt cells.