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REGULATORY MECHANISM OF THE EPITHELIAL Na^+ CHANNEL (ENaC) BY CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR)

囊性纤维化跨膜电导调节器 (CFTR) 对上皮 Na^ 通道 (ENaC) 的调节机制

基本信息

批准号：
12670035
负责人：
ISHIKAWA Toru
金额：
$ 2.18万
依托单位：
HOKKAIDO 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

项目摘要

I have previously found that cytosolic ATP regulates activity of the rat epithelial Na1 channel (αβγ-rENaC) heterologously expressed in NIH-3T3 cells. Using both patch-clamp and biochemical techniques, I have now shown that the ATP regulation of rENaC is also seen in rENaC stably expressed.in MDCK epithelial cells and that the regulation is likely mediated through a non-hydrolytic nucleotide binding to α-rENaC. In order to identify the ATP binding sites I have generated cell lines expressing mutant α-rENaCs.ENaC is known to be regulated by cystic fibrosis transmembrane conductance regulator (CFTR), which functions as a cAMP-regulated, ATP-dependent Cl^-channel Since CFTR is localized in the apical membrane of epithelial cells and also possesses ATPase activity necessary for Cr conductance, I hypothesized that ENaC regulation by CFTR may be also mediated by a mechanism through which cytosolic ATP regulates ENaC. To begin with, I first characterized electrophysiological properties of rENaC in NIH3T3 cells coexpressing rENaC and CFTR using patch-clamp techniques. Electrophysiological properties of rENaC such as ion selectivity, amiloride-sensitivity were similar to those in NIH3T3 cells expressing rENaC-alone. I also showed that CFTR activity was seen in NIH3T3 cells coexpressing rENaC and CFTR, but not in the cells expressing rENaC alone. In order to study functional interaction ofrENaC and CFTR in native epithelial cells, native rENaC currents in freshly isolated rat colonic surface epithelial cells, where rENaC was first cloned and where CFTR has been known to be expressed, were first functionally characterized using whole-cell patch-clamp techniques. Electrophysiological properties such as amiloride-sensitivity, ion selectivity of native amiloride-sensitive currents were strikingly similar to those of the cloned rENaC expressed in MDCK and NIH-3T3 cells.By using these experimental systems, I am going to systematically test the hypothesis mentioned above.

我以前发现，胞浆三磷酸腺苷调节异源表达的大鼠上皮细胞NA1通道(αβγ-rENaC)的活性。使用膜片钳和生化技术，我现在已经证明，rENaC的三磷酸腺苷调节也在rENaC中稳定表达。在mDCK上皮细胞中，这种调节可能是通过与α-rENaC的非水解性核苷酸结合而介导的。为了确定三磷酸腺苷结合位点，我建立了表达突变体α-rENaC的细胞系。ENaC是由囊性纤维化跨膜电导调节器调节的，由于囊性纤维化跨膜电导调节器定位于上皮细胞顶膜，也具有肌电导所必需的ATPase活性，因此我推测囊性纤维化跨膜电导调节器对ENaC的调节也可能是通过胞浆三磷酸腺苷调节ENaC的机制来调节的。首先，我首先利用膜片钳技术在共表达rENaC和CFTR的NIH3T3细胞中研究了rENaC的电生理特性。RENaC的离子选择性、阿米洛利敏感性等电生理特性与单独表达rENaC的NIH3T3细胞相似。我还发现CFTR活性在共表达rENaC和CFTR的NIH3T3细胞中可见，但在单独表达rENaC的细胞中未见。为了研究rENaC和CFTR在天然上皮细胞中的功能相互作用，首先用全细胞膜片钳技术研究了rENaC在新鲜分离的大鼠结肠表面上皮细胞中的天然rENaC电流，其中rENaC是首次被克隆的，CFTR也是已知表达的地方。天然阿米洛利敏感电流对阿米洛利的敏感性、离子选择性等电生理特性与在MDCK和NIH-3T3细胞中表达的克隆的rENaC非常相似。通过使用这些实验系统，我将系统地检验上述假设。