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Molecular biological examination of Calcium-sensitive Chloride Channel expressed in the kidney

肾脏表达的钙敏感氯离子通道的分子生物学检查

基本信息

批准号：
07671242
负责人：
SAKAMOTO Hisato
金额：
$ 1.34万
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 (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

A new chloride channel that belongs to ClC chloride channel family has been identified from rat kidney by a sequence homology-based strategy and designated as ClC-5, ClC-5 cDNA encodes a polypeptide of 746 amino acids, which is indicated by hydrophobicity analysis to have structural features that are common of the ClC family. Northern blot analysis of rat tissues showed that ClC-5 mRNA was predominantly expressed in the kidney and colon. According to the phylogenic analysis ClC-5 chloride channel can be classified into a subfamily : the ClC-3/ClC-4/ClC-5 chloride channels. To characterized the functional properties of ClC-5 by patch-clamp technique, we established the stably transfected cultured cells line using intranuclear microinjection technique. The transfected cells induced out-wardly rectifying chloride currents (ORCC) on whole cell configuration as same as that of ClC-3. In addition, the properties of ClC-5 channel have been examined at the level of single channel using the inside-out patch technique. In addition, when we have identified the ClC-5 from rat kidney, human ClCN5 cDNA which was counterpart of rat ClC-5 has been independently reported as a candidate gene for three different inherited kidney diseases (Dent's disease, X-linked recessive nephrolithiasis, and X-linked recessive hypophosphataemic rickets). In the clinical approach, the mutations in ClCN5 gene were also identified from three patients associated with idiopathic low-molecular-weight proteinuria. However, it is not clear how loss of this renal chloride channel can result in diverse renal dysfunction in these disease. Now, ClC-5 has attracted a special interest in relation to these hereditary renal disease. To examine the molecular structure and functional properties of ClC-5 will provide an important insight into these pathogenesis of these inherited disease associated with common molecular abnormalities of ClCN5 gene.

通过基于序列同源性的策略，从大鼠肾脏中鉴定出属于ClC氯离子通道家族的一个新的氯离子通道，并将其命名为ClC-5，ClC-5 cDNA编码746个氨基酸的多肽，疏水性分析表明其具有ClC家族共有的结构特征。大鼠组织的 Northern 印迹分析表明 ClC-5 mRNA 主要在肾脏和结肠中表达。根据系统发育分析，ClC-5氯离子通道可分为一个亚家族：ClC-3/ClC-4/ClC-5氯离子通道。为了通过膜片钳技术表征ClC-5的功能特性，我们利用核内显微注射技术建立了稳定转染的培养细胞系。转染细胞在整个细胞结构上诱导外向整流氯电流(ORCC)，与ClC-3相同。此外，还使用由内而外的贴片技术在单通道水平上检查了 ClC-5 通道的特性。此外，当我们从大鼠肾脏中鉴定出 ClC-5 时，与大鼠 ClC-5 相对应的人 ClCN5 cDNA 已被独立报道为三种不同遗传性肾脏疾病（登特氏病、X 连锁隐性肾结石和 X 连锁隐性低磷血症性佝偻病）的候选基因。在临床方法中，还从三名与特发性低分子量蛋白尿相关的患者中鉴定出 ClCN5 基因突变。然而，尚不清楚这种肾氯离子通道的丧失如何导致这些疾病中的多种肾功能障碍。现在，ClC-5 引起了与这些遗传性肾病相关的特殊兴趣。检查 ClC-5 的分子结构和功能特性将为了解这些与 ClCN5 基因常见分子异常相关的遗传性疾病的发病机制提供重要的见解。