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Regulation of CLC chloride channels by their beta-subunits

CLC 氯离子通道的 β 亚基调节

基本信息

批准号：
16390241
负责人：
UCHIDA Shinichi
金额：
$ 9.15万
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：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

We investigated intracellular sorting mechanisms of renal CLC chloride channels by their beta-subunit barttin. To solve this issue, we focused on the sorting mechanisms of barttin itself and how CLC-K and barttin were interacted.1. Identification of CLC-K and barttin inter-acting domains.We investigated how CLC-K and barttin form hetero-oligomer by generating several truncated mutants of CLC-K and barttin and performing co-immunoprecipitation assay. We found that barttin binds to CLC-K on its domain B and C, and domain J and K. Two membrane spanning regions of barttin are necessary for its binding to CLC-K.2. Trial to rescue a disease-causing mutant barttin by drugs.Barttin recruits CLC-K to the plasma membranes. R8L barttin, a disease-causing mutant, is retained in endoplasmic reticulum (ER). However, it can bind to CLC-K. Accordingly, CLC-K is also retained in ER. This is a major mechanism of Bartter syndrome caused by barttin mutaions. In order to release R8L from ER and make it translocate to plasma membranes, several drugs including chemical chaperones and curcumin were tested using R8L stably expressing MDCK cells. Curcumin (10uM), glycerol (1M), and DMSO (2%) were found to be effective in releasing R8L from ER. To test this potential therapeutic strategy in vivo, we prepared a targeting construct for making R8L knock-in mouse.3. Identification of basolateral sorting signal of barttin.Basolateral sorting signal of barttin was determined by expressing several truncated mutants of barttin in MDCK cells. A 15-20 amino acid stretch in the carboxy cytoplasmic region of barttin was necessary for its basolateral sorting, which may be a new type of basolateral sorting signal.

我们研究了肾小细胞肺癌氯离子通道的细胞内分选机制。为了解决这一问题，我们重点研究了键合蛋白本身的分选机制以及CLC-K与键合蛋白如何相互作用。CLC-K与巴丁蛋白相互作用域的鉴定。我们通过产生CLC-K和巴丁的几个截短突变体并进行共免疫沉淀实验来研究CLC-K和巴丁如何形成异聚物。我们发现，巴丁蛋白在其结构域B和C以及结构域J和k上与CLC-K结合。巴丁蛋白的两个跨膜区域是其与CLC-K - 2结合所必需的。尝试用药物拯救一种致病突变的巴丁蛋白。bartin将CLC-K招募到质膜上。致病突变体R8L巴丁蛋白保留在内质网（ER）中。然而，它可以与CLC-K结合。因此，CLC-K也保留在ER中。这是巴特尔蛋白突变引起巴特尔综合征的主要机制。为了将R8L从内质网释放并转运至质膜，我们在稳定表达R8L的MDCK细胞上测试了化学伴侣和姜黄素等几种药物。姜黄素（10uM）、甘油（1M）和DMSO（2%）能有效地从内质网释放R8L。为了在体内测试这种潜在的治疗策略，我们准备了一个靶向构建体来制造R8L敲入小鼠。巴丁碱基侧分选信号的识别。通过在MDCK细胞中表达几个截断的巴丁突变体来确定巴丁的基底外侧分选信号。巴丁蛋白的羧基细胞质区有一个15-20个氨基酸的延伸，这可能是一种新的基侧分选信号。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Regulation of apical localization of the thiazide-sensitive NaCl cotransporter by WNK4 in polarized epithelial cells.

DOI：
10.1016/j.bbrc.2005.02.172
发表时间：
2005-05
期刊：
Biochemical and biophysical research communications
影响因子：
3.1
作者：
S. Yang;K. Yamauchi;T. Rai;Atsushi Hiyama;E. Sohara;Tatsunori Suzuki;T. Itoh;Shin Suda;S. Sasaki;S. Uchida
通讯作者：
S. Yang;K. Yamauchi;T. Rai;Atsushi Hiyama;E. Sohara;Tatsunori Suzuki;T. Itoh;Shin Suda;S. Sasaki;S. Uchida

Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins.

DOI：
10.1073/pnas.0306924101
发表时间：
2004-03
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
K. Yamauchi;T. Rai;Katsuki Kobayashi;E. Sohara;Tatsunori Suzuki;T. Itoh;Shin Suda;Atsushi Hayama;S. Sasaki;S. Uchida
通讯作者：
K. Yamauchi;T. Rai;Katsuki Kobayashi;E. Sohara;Tatsunori Suzuki;T. Itoh;Shin Suda;Atsushi Hayama;S. Sasaki;S. Uchida

Function of chloride channels in the kidney.

肾脏中氯离子通道的功能。

DOI：
发表时间：
2005
期刊：
Annu Rev Physiol 67
影响因子：
0
作者：
Uchida S et al.
通讯作者：
Uchida S et al.

Intracellular localization of ClC chloride channels and their ability to hetero-oligomers

ClC 氯离子通道的细胞内定位及其异源寡聚物的能力

DOI：
发表时间：
2006
期刊：
J Cell Physiol 206
影响因子：
0
作者：
Tagawa H;Kizuka Y;Ikeda T;Itoh S;Kawasaki N;Kurihara H;Onozato M-L;Tojo A;Sakai T;Kawasaki T;Oka S;Yamamoto Y et al.;栗原秀剛;Kamitani T;栗原秀剛;Suzuki T et al.
通讯作者：
Suzuki T et al.

WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1