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

我们克隆了C1C-3B，一种新的C1C-3（C1C-3A）的选择性剪接变体，主要在上皮细胞中表达。CIC-3B具有与C1C-3A不同的稍长的C末端，并且含有用于结合上皮特异性支架蛋白EBP 50的第二PDZ结构域的共有基序。体外和体内结合测定均证明C1C-3B和EBP 50之间的相互作用。单独转染C1C-3B的C127小鼠乳腺上皮细胞在胞质区域显示C1C-3B的弥漫性免疫反应性。相反，当EBP50与C1C-3B共转染时，C1C-3B的强免疫反应性出现在膜皱褶的前缘。膜片钳实验显示C1C-3B和EBP 50共转染导致C127细胞膜皱褶前缘的外向整流C1通道（ORCC）活性显著增加。C1C-3B诱导的ORCC的电生理特性与天然上皮细胞中描述的ORCC的电生理特性相似。当囊性纤维化跨膜传导调节因子（CFTR）与C1C-3B和EBP 50共转染时，C1C-3B依赖的ORCCs通过蛋白激酶A依赖的途径被激活。这些发现表明C1C-3B本身是CFTR调节的ORCC分子或其激活剂。由于突变的CFTR对ORCC的PKA依赖性激活在CF上皮中是有缺陷的，因此ORCC及其通过CFTR的调节已经得到了很好的研究。正常的ORCC活性可以通过不依赖于CFTR和PKA的方式来诱导，例如去极化、细胞外ATP和src样激酶，所有这些都代表了治疗CF的潜在治疗靶点。在本研究中，我们证明C1C-3B和EBP 50的共转染刺激由CFTR调节的ORCC活性。这些发现对于更好地理解上皮C1通道和CF的病理生理学是重要的。

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.