Studies on the physiological roles of intracellular OLO chloride channels

细胞内OLO氯通道的生理作用研究

• 批准号: 14370316
• 负责人: UCHIDA Shinichi
• 金额: $ 8.9万
• 依托单位: Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370316/
• 关键词: CLC chloride channel; intracellular sorting; Golgi; MDCK cells; Bartter syndrome; immunoprecipitation; エンドソーム; ライソゾーム; neuronal ceroid lipofuscinosis; ノックアウトマウス; トランスジェニックマウス; F1F0-ATPase; Iysosomal storage disease; クロライドチャネル; 水チャネル

1) We generated transgenic mice expressing GFP under the control of CLC-KB gene promoter. It clearly determined the sites of intrarenal expression of CLC-KB.2) We generated CLC-3 KO mice, and found that CLC-3 is functioning in intracellular acidic organelles, and that it is involved in protein-degradation pathways.3) We successfully isolated fish CLC-K channel. This provided us information on the ancient role of CLC-K channels.4) We determined the molecular pathogenesis of Bartter's syndrome by barttin mutations in mammalian cells. In immunocytochemistry, CLC-K2 was retained in the Golgi in the absence of barttin expression, but delivered to the plasma membrane when barttin was present. Bartlin was co-precipitated with CLC-K2, suggesting a protein-protein interaction. Disease-causing mutant barttins, especially R8L, were retained intracellularly, but their binding ability to CLC-K2 was preserved. This led to a retention of CLC-K2 in intracellular organelles with barttin, and a loss of plasma membrane localization.

1)在CLC-KB基因启动子的控制下，构建表达GFP的转基因小鼠。明确了CLC-KB在肾内的表达位点。2)我们生成了CLC-3 KO小鼠，发现CLC-3在细胞内酸性细胞器中起作用，并参与蛋白质降解途径。3)成功分离了鱼类的CLC-K通道。这为我们提供了有关CLC-K通道古代作用的信息。4)我们通过哺乳动物细胞中的巴丁蛋白突变确定了巴氏综合征的分子发病机制。在免疫细胞化学中，当没有巴丁表达时，CLC-K2保留在高尔基体中，但当有巴丁存在时，CLC-K2被传递到质膜上。Bartlin与CLC-K2共沉淀，提示存在蛋白-蛋白相互作用。致病突变的巴丁蛋白，尤其是R8L，在细胞内被保留，但它们与CLC-K2的结合能力被保留。这导致CLC-K2保留在细胞内的细胞器与巴丁，并失去质膜定位。

项目成果

Katsuki Kobayashi et al.: "Human CL C-KB gene promoter drives the EGFP expression in the specific distal nephron segments and inner ear."J.Am.Soc.Nephrol.. 13. 1992-1998 (2002)

Katsuki Kobayashi 等人：“人类 CL C-KB 基因启动子驱动特定远端肾单位段和内耳中的 EGFP 表达。”J.Am.Soc.Nephrol.. 13. 1992-1998 (2002)

Teiko Ohashi: "Intracellular mislocalization of mutant podocin and correction by chemical chaperones."Histochem Cell Biol. 119. 257-264 (2003)

Teiko Ohashi：“突变体 podocin 的细胞内错误定位和化学伴侣的校正。”组织化学细胞生物学。

KATSUKI KOBAYASHI: "Human CLC-KB gene promotion derives the EGFP expression in the specific distal nephron segments and inner ear"J.Am.Soc.Nephrol.. 13. 1992-1998 (2002)

KATSUKI KOBAYASHI：“人类 CLC-KB 基因促进导致特定远端肾单位段和内耳中的 EGFP 表达”J.Am.Soc.Nephrol.. 13. 1992-1998 (2002)

ATSUSHI HAYAMA: "Molecular mechanisms of Batter syndrome caused by mutations in the BSNO gene"Histochem.Cell.Biol.. 119. 485-493 (2003)

ATSUSHI HAYAMA：“BSNO 基因突变引起的巴特综合征的分子机制”Histochem.Cell.Biol.. 119. 485-493 (2003)

Hirofumi Maehara et al.: "Expression of CLC-KB gene promoter in the mouse cochlea."Neuro Report. 14. 1571-1573 (2003)

Hirofumi Maehara 等人：“CLC-KB 基因启动子在小鼠耳蜗中的表达。”神经报告。