Mohammad-Panah R, Gyomorey K, Rommens J, Choudhury M, Li C, Wang Y, Bear C E
Programme in Cell Biology and Genetics at the Hospital for Sick Children and the Departments of Physiology and Molecular Genetics at the University of Toronto, Toronto, M5G 1X8 Ontario, Canada.
J Biol Chem. 2001 Mar 16;276(11):8306-13. doi: 10.1074/jbc.M006764200. Epub 2000 Nov 28.
It has been previously determined that ClC-2, a member of the ClC chloride channel superfamily, is expressed in certain epithelial tissues. These findings fueled speculation that ClC-2 can compensate for impaired chloride transport in epithelial tissues affected by cystic fibrosis and lacking the cystic fibrosis transmembrane conductance regulator. However, direct evidence linking ClC-2 channel expression to epithelial chloride secretion was lacking. In the present studies, we show that ClC-2 transcripts and protein are present endogenously in the Caco-2 cell line, a cell line that models the human small intestine. Using an antisense strategy we show that ClC-2 contributes to native chloride currents in Caco-2 cells measured by patch clamp electrophysiology. Antisense ClC-2-transfected monolayers of Caco-2 cells exhibited less chloride secretion (monitored as iodide efflux) than did mock transfected monolayers, providing the first direct molecular evidence that ClC-2 can contribute to chloride secretion by the human intestinal epithelium. Further, examination of ClC-2 localization by confocal microscopy revealed that ClC-2 contributes to secretion from a unique location in this epithelium, from the apical aspect of the tight junction complex. Hence, these studies provide the necessary rationale for considering ClC-2 as a possible therapeutic target for diseases affecting intestinal chloride secretion such as cystic fibrosis.
先前已经确定，ClC氯化物通道超家族的成员ClC-2在某些上皮组织中表达。这些发现引发了一种推测，即ClC-2可以补偿受囊性纤维化影响且缺乏囊性纤维化跨膜传导调节因子的上皮组织中受损的氯化物转运。然而，缺乏将ClC-2通道表达与上皮氯化物分泌联系起来的直接证据。在本研究中，我们表明ClC-2转录本和蛋白内源性存在于Caco-2细胞系中，该细胞系可模拟人类小肠。使用反义策略，我们表明ClC-2对通过膜片钳电生理学测量的Caco-2细胞中的天然氯化物电流有贡献。与空载体转染的单层细胞相比，反义ClC-2转染的Caco-2细胞单层表现出较少的氯化物分泌（以碘化物外流监测），这提供了首个直接的分子证据，证明ClC-2可促进人肠上皮细胞的氯化物分泌。此外，通过共聚焦显微镜检查ClC-2的定位发现，ClC-2在该上皮细胞紧密连接复合体顶端的独特位置对分泌有贡献。因此，这些研究为将ClC-2视为影响肠氯化物分泌的疾病（如囊性纤维化）的可能治疗靶点提供了必要的理论依据。
