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Intracellular trafficking mechanisms of renal C1C and AQP channels

肾C1C和AQP通道的细胞内转运机制

基本信息

批准号：
12144204
负责人：
UCHIDA Shinichi
金额：
$ 32.77万
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 on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

项目摘要

We investigated intracellular sorting mechanisms of renal CLC chloride-channels and AQP water-channels, especially focusing on the disease-causing mutants of these channels.1. Molecular pathogenesis of autosomal-dominant type nephrogenic diabetes insipidus (AD-NDI) in AQP2. We found five families of AD-NDI. Sequencing of AQP2-gene revealed the heterozygous frame-shift mutations in all patients, resulting in addition of 61 amino-acids to carboxy-terminus of AQP2. In contrast to the apical localization of wild-type AQP2 expressed in MDCK cells, the disease-causing mutants basolaterally distributed. Furthermore, the mutants recruited the wild-type AQP2 to the basolateral membrane when co-expressed, showing dominant-negative effect. We also found that a di-luecine motif in the mutant was responsible for the basolateral sorting. To confirm whether this mechanism worked in vivo, we generated by gene-targeting a knock-in mouse expressing the mutant AQP2. As expected, the mice showed AD-NDI. A … More pical localization of wild-type AQP2 was significantly impaired by the mutant AQP2-expression. Further analysis of this mouse model will help to clarify the molecular mechanisms of AD-NDI and to develop strategy for treatments of AD-NDI.2. Molecular mechanisms of Bartter syndrome caused by mutations in the BSND-gene. The BSND-gene encodes barttin, which functions as beta-subunit of Cl-channel C1C-K. We determined intracellular localization of C1C-K2 with or without co-expressing barttin in MDCK cells. Barttin clearly recruited C1C-K2 to the plasma membranes. R8L barttin, a disease-causing mutant, was retained in endoplasmic reticulum (ER). However, it could bind C1C-K2. Accordingly, C1C-K2 was also retained in ER. This might be a major mechanism of Bartter syndrome caused by barttin mutaions.3. Chloride-shunt theory as a cause of pseudohypoaldosteronism type II (PHAII). Mutaions of WNK-kinases were identified to be responsible for PHAII. However, its molecular pathogenesis was not determined. We demonstrated that a disease-causing WNK4-mutant increased paracellular chloride-permeability and claudin-phosphorylation. Less

我们研究了肾脏ClC氯通道和水通道的细胞内分选机制，特别是这些通道的致病突变。AQP2常染色体显性遗传型肾源性尿崩症(AD-NDI)的分子发病机制我们发现了五个AD-NDI家系。对AQP2基因进行测序发现，所有患者都存在杂合性移码突变，导致AQP2的羧基末端增加了61个氨基酸。与野生型AQP2在MDCK细胞中表达的顶端定位相反，致病突变体基本呈基线分布。此外，当共表达时，突变体将野生型AQP2募集到基侧膜上，表现出显性-负效应。我们还发现，突变体中的一个双侧基序负责碱侧排序。为了证实这一机制在体内是否起作用，我们通过对表达突变AQP2的敲入小鼠进行基因打靶产生了这种机制。不出所料，这些小鼠出现了AD-NDI。A…突变型AQP2的表达显著削弱了野生型AQP2更常见的定位。对这一小鼠模型的进一步分析将有助于阐明AD-NDI的分子机制，并为AD-NDI的治疗制定策略。BSND基因突变引起巴特综合征的分子机制BSND-基因编码Barttin，其功能是氯离子通道C1C-K的β-亚基。我们确定了在MDCK细胞中有或没有共表达Barttin的C1C-K2的细胞内定位。Barttin清楚地将C1C-K2招募到质膜上。致病突变体R8L barttin保留在内质网(ER)中。然而，它可以结合C1C-K2。因此，C1C-K2也保留在内质网中。这可能是Barttin突变导致巴特综合征的主要机制。氯分流理论是假性醛固酮增多症II型(PHAII)的原因。WNK-激酶突变被确定为PHAII的致病因素。然而，其分子发病机制尚未确定。我们证明了致病的WNK4突变体增加了细胞旁氯离子的通透性和Claudin的磷酸化。较少

项目成果

期刊论文数量（211）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Kruppel-like factor, a novel zinc finger repressor

DOI：
10.1128/mcb.20.19.7319-7331.2000
发表时间：
2000-10-01
期刊：
MOLECULAR AND CELLULAR BIOLOGY
影响因子：
5.3
作者：
Uchida, S;Tanaka, Y;Marumo, F
通讯作者：
Marumo, F

Isolation and characterization of the human CLC-5 chloride channel gene promoter

DOI：
10.1016/s0378-1119(00)00493-5
发表时间：
2000-12-31
期刊：
GENE
影响因子：
3.5
作者：
Hayama, A;Uchida, S;Marumo, F
通讯作者：
Marumo, F

Expression of CLC-KB gene promoter in the mouse cochlea.

CLC-KB基因启动子在小鼠耳蜗中的表达。

DOI：
发表时间：
2003
期刊：
NeuroReport 14(12)
影响因子：
0
作者：
Minamisawa S;Oshikawa J;Takeshima H;Hoshijima M;Wang Y;Chien KR;Ishikawa Y;Matsuoka R;Maehara H et al.
通讯作者：
Maehara H et al.

Kidney-specific chloride channel, OmC1C-K, predominantly expressed in the diluting segment of freshwater-adapted tilapia kidney.

肾脏特异性氯离子通道 OmC1C-K 主要在淡水适应罗非鱼肾脏的稀释段中表达。