权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Roles of cloned chloride channels in kidney chloride transport

克隆氯离子通道在肾脏氯离子转运中的作用

基本信息

批准号：
07671241
负责人：
UCHIDA Shinichi
金额：
$ 1.54万
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

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671241/
关键词：
chloride channel immunohistochemistry human gene FISH method イオントランスポート尿濃縮腎臓

项目摘要

To determine the intrarenal localization of three chloride channels isolated by us, we prepared antisera for rat ClC-K1, -K2, -3 using synthetic peptide as antigens. We obtained specific antisera to ClC-K1 and ClC-K2. Using these antisera, we performed immunohistochemical study to localize the intrarenal localizaton of ClC-K1 and ClC-K2. ClC-K1 is present in the both apical and basolateral plasma membranes of the thin ascending limb of Henle's loop, and ClC-K2 is present in the apical plasma membrane of the connecting tubules. These findings will shed light on the physiological roles if ClC-K1 and -K2 in the kidney.Next, we studied structure- function relationship of ClC-K1 and ClC-3. The in vitro mutations of positively charged amino acids in the first extracellular loop of ClC-K1 into negatively charged amino acids greatly enhanced the chloride current expressed in Xenopus oocytes, suggesting the importance of this region. Further studies using chimera chloride channels of ClC-K1 and -K2 are in progress. As for ClC-3, a stably expressing cell line was established in CHO cells, and patch clamp analysis revealed the Ca-regulated nature of ClC-3. Finally, we isolated human ClC-K1 and -K2 cDNAs to study the involvements of these genes in certain human kidney diseases. Using these cDNA,we further isolated human ClC-K1 and -K2 genes and characterized the gene structure including exon-intron structures. We prepared PCR primer sets to cover all exons and exon-intron boundaries and could established the system to determine the sequences of ClC-K2 gene from patients by direct sequencing. Also, we determined the chromosomal localization of ClC-K1, -K2 and -3 by FISH method.

为了确定我们分离的三种氯离子通道的肾内定位，我们使用合成肽作为抗原制备了大鼠ClC-K1、-K2、-3的抗血清。我们获得了针对 ClC-K1 和 ClC-K2 的特异性抗血清。使用这些抗血清，我们进行了免疫组织化学研究以定位 ClC-K1 和 ClC-K2 的肾内定位。 ClC-K1 存在于亨利氏袢薄升肢的顶端和基底外侧质膜中，ClC-K2 存在于连接小管的顶端质膜中。这些发现将阐明ClC-K1和-K2在肾脏中的生理作用。接下来，我们研究了ClC-K1和ClC-3的结构-功能关系。 ClC-K1第一个细胞外环中带正电的氨基酸在体外突变为带负电的氨基酸，大大增强了非洲爪蟾卵母细胞中表达的氯电流，表明该区域的重要性。使用 ClC-K1 和 -K2 的嵌合氯化物通道的进一步研究正在进行中。对于ClC-3，在CHO细胞中建立了稳定表达的细胞系，膜片钳分析揭示了ClC-3的Ca2+调节性质。最后，我们分离了人类 ClC-K1 和 -K2 cDNA，以研究这些基因与某些人类肾脏疾病的关系。利用这些cDNA，我们进一步分离了人类ClC-K1和-K2基因，并表征了基因结构，包括外显子-内含子结构。我们制备了覆盖所有外显子和外显子-内含子边界的PCR引物组，并建立了通过直接测序确定患者ClC-K2基因序列的系统。此外，我们还通过FISH方法确定了ClC-K1、-K2和-3的染色体定位。