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

我之前发现细胞质ATP调节NIH-3T3细胞中异源表达的大鼠上皮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ATP对rENaC的调节也见于rENaC的稳定表达。这种调节可能是通过与α-rENaC结合的非水解核苷酸介导的。为了鉴定ATP结合位点，我产生了表达突变α-rENaCs的细胞系。众所周知，ENaC受囊性纤维化跨膜传导调节剂（CFTR）的调控，CFTR是camp调控的、ATP依赖的Cl^通道。由于CFTR位于上皮细胞的顶膜，并且具有Cr传导所必需的ATP酶活性，我假设CFTR对ENaC的调控也可能通过胞质ATP调节ENaC的机制来介导。首先，我使用膜片钳技术在共表达rENaC和CFTR的NIH3T3细胞中表征了rENaC的电生理特性。rENaC的电生理特性如离子选择性、阿米洛胺敏感性等与单独表达rENaC的NIH3T3细胞相似。我还表明，在共表达rENaC和CFTR的NIH3T3细胞中可以看到CFTR活性，而在单独表达rENaC的细胞中则没有。为了研究天然上皮细胞中rENaC和CFTR的功能相互作用，首先使用全细胞膜片钳技术对新鲜分离的大鼠结肠表面上皮细胞中的天然rENaC电流进行了功能表征，rENaC是首次克隆的，CFTR是已知表达的。天然amiloride敏感电流的电生理特性，如amiloride敏感性，离子选择性与MDCK和NIH-3T3细胞中表达的克隆rENaC惊人地相似。通过使用这些实验系统，我将系统地检验上述假设。

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.