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Cloning and functional expression of a novel C1 channel that interacts with EBP50

与 EBP50 相互作用的新型 C1 通道的克隆和功能表达

基本信息

批准号：
12670083
负责人：
OGURA Takehiko
金额：
$ 2.05万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670083/
关键词：
C1C-3 EBP50 PDZ DOMAIN CFTR ORCC ClC-3 Cl^-チャネル

项目摘要

We have cloned C1C-3B, a novel alternative splicing variant of C1C-3 (C1C-3A) that is expressed predominantly in epithelial cells. CIC-3B has a different, slightly longer C-terminal end than C1C-3A, and contains a consensus motif for binding to ,the second PDZ domain of the epithelium-specific scaffolding protein EBP50. Both in vitro and in vivo binding assays demonstrate interaction between C1C-3B and EBP50. C127 mouse mammary epithelial cells transfected with C1C-3B alone showed diffuse immunoreactivity for C1C-3B in the cytoplasmic region. In contrast, when EBP50 was co-transfected with C1C-3B, strong immunoreactivity for C1C-3B appeared at the leading edges of membrane ruffles. Patch-clamp experiments revealed that co-transfectipn of C1C-3B and EBP50 resulted in a remarkable increase in outwardly rectifying C1-channel (ORCC) activities at the leading edges of membrane ruffles in C127 cells. The electrophysiological properties of the C1C-3B-induced ORCCs are similar to those of ORCCs described in native epithelial cells. When cystic fibrosis transmembrane conductance regulator (CFTR) was co-transfected with C1C-3B and EBP50, C1C-3B-dependent ORCCs were activated via the protein kinase A-dependent pathway. These findings indicate that C1C-3B is itself a CFTR-regulated ORCC molecule or its activator. Because PKA-dependent activation of the ORCC by mutated CFTR is defective in CF epithelia, ORCCs and their regulation by CFTR have been well studied. Normal ORCC activities can be induced by means independent of CFTR and PKA, such as depolarization, extracellular ATP, and an src-like kinase, all of which represent potential therapeutic targets for the treatment of CF. In the present study, we demonstrate that co-transfection of C1C-3B and EBP50 stimulates ORCC activity which is regulated by CFTR. These findings are important to better understand both epithelial C1- channels and the pathophysiology of CF.

我们克隆了C1C-3B，它是C1C-3(C1C-3A)的一个新的选择性剪接变异体，主要在上皮细胞中表达。CIC-3B有一个不同于C1C-3A的略长于C1C-3A的C末端，并包含一个与上皮特异性支架蛋白EBP50的第二个PDZ结构域结合的共识基序。体外和体内结合试验均证明C1C-3B与EBP50之间存在相互作用。单独转染C1C-3B的C127小鼠乳腺上皮细胞胞浆区呈弥漫性免疫反应。相反，当EBP50与C1C-3B共转染时，C1C-3B在膜皱纹的前沿出现强烈的免疫反应。膜片钳实验表明，C1C-3B和EBP50共转染后，C127细胞膜褶皱前沿的外向整流C1通道(ORCC)活性显著增加。C1C-3B诱导的ORCCs的电生理特性与天然上皮细胞中描述的ORCCs相似。当囊性纤维化跨膜电导调节因子(CFTR)与C1C-3B和EBP50共转染时，C1C-3B依赖的ORCC通过依赖蛋白激酶A的途径被激活。这些发现表明，C1C-3B本身就是CFTR调控的ORCC分子或其激活剂。由于突变的CFTR依赖于PKA激活的ORCC在CF上皮细胞中是有缺陷的，ORCCs及其调控已经得到了很好的研究。正常的ORCC活性可以通过不依赖CFTR和PKA的方法来诱导，如去极化、细胞外ATP和src样激酶，所有这些都是治疗CF的潜在治疗靶点。在本研究中，我们证明了C1C-3B和EBP50的共转染可以刺激受CFTR调控的ORCC活性。这些发现对于更好地理解上皮性c1通道和cf的病理生理学具有重要意义。