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Structure and function of novel pumps and channels in gastrointestinal tract

胃肠道新型泵和通道的结构和功能

基本信息

批准号：
13307063
负责人：
TAKEGUCHI Noriaki
金额：
$ 24.29万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13307063/
关键词：
gastric proton pump gastric acid secretion gastric flippase proton pump inhibitor chloride channel cytoprotection interleukin nitric oxide 大腸プロトンポンプ胃フリツパーゼ

项目摘要

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.

1.成功构建了稳定表达胃质子泵α-和β-亚单位的细胞系（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。2.在表达胃质子泵的HEK-293细胞中，我们发现M6段中对质子泵功能有重要作用的氨基酸残基仅位于α-螺旋结构的一侧。我们还发现，β亚基胞外域中的所有三个二硫键对于α-β组装、质子泵活性、α和β亚基的稳定性，3.我们成功地建立了表达胃质子泵mRNA的条件永生化胃粘膜细胞系，该细胞系来自携带温度敏感猿猴病毒40大T-IL-1 β与IL-1受体结合，通过G蛋白介导的Rho/Rho激酶依赖的O_2 ^-产生抑制基底外侧管家Cl ^-通道。CLCA 1不是胃管家Cl ^-通道的亚基。5.我们发现NO依赖性Cl ^-通道激活的新机制。在这一机制中，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。