Structure and function of novel pumps and channels in gastrointestinal tract

1.成功构建了稳定表达胃质子泵α-和β-亚基的细胞系(极化LLC-PK1细胞)。2.在用表达胃质子泵的HEK293细胞进行的研究中，我们发现M6片段中与质子泵功能有关的氨基酸残基仅定位在α-螺旋结构的一侧。我们还发现，β亚基胞外区的三个二硫键对α-β的组装、质子泵活性、α和β亚基的稳定性以及α亚基的细胞表面传递都是必需的。3.我们成功地从携带温度敏感的猴病毒40大T抗原的转基因小鼠中建立了表达胃质子泵基因的条件永生化胃粘膜细胞系。4.我们发现IL-1β通过G蛋白介导的RHO/Rho-K依赖的底侧管家氯通道的产生而与兔胃壁细胞的IL-1受体结合。5.我们发现大鼠结肠粘膜中NO依赖的氯离子通道激活的新机制。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氯离子通道。

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.