INVESTIGATION OF ALTERNATE CHLORIDE CHANNELS

囊性纤维化是一种最常见的致死性常染色体隐性遗传病 高加索人的精神障碍。它是由CF的突变引起的 跨膜电导调节器(CFTR)。这种蛋白质的功能是 功能正常的CFTR导致氯、钠和水的异常运动 穿过肺、胰腺和肺上皮细胞的顶膜 输精管综合征。其结果是脱水和增加粘度 粘液分泌物导致梗阻、炎症和进行性 破坏受影响的器官。其他离子通道的激活， 尤其是根尖膜上存在的氯通道。 CF患者的上皮细胞可能绕过氯化物中的缺陷 由cftr突变引起的传导。氯离子通道随时间变化 已在呼吸道上皮细胞中鉴定出其特性。我们最近做了 获得了预测氨基酸序列高度同源的人cDNA1 与电压和体积调节的氯离子通道的相似程度 大鼠(ClC-2)。人类序列在多种组织中表达 包括肺和胰腺以及众多的呼吸道上皮细胞系 来源于慢性萎缩性胃炎患者。这个项目的总体目标是 确定是否可以操纵人类ClC-2氯通道 通过研究改善CF电池的氯离子传导缺陷 它的功能特性和蜂窝位置。这将是 通过追求以下目标来实现： 1)完成hClC-2基因5‘端的克隆及鉴定 通过确定转录的起始点来确定它是全长的。 2)研究非洲爪哇卵母细胞的氯离子电流 哺乳动物293细胞瞬时表达全长hClC-2基因。 3)确定人工合成的hClC-2的细胞定位 极化上皮细胞(MDCK)稳定表达表位(FLAG) 使用顶端和基底侧面细胞膜标记物。 4)确定上皮细胞中hClC-2编码的氯离子通道 通过反义技术减少内源性hClC-2的表达。

Cystic fibrosis (CF) is one of the most common lethal autosomal recessive disorders in Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR). This protein functions as functional CFTR causes abnormal chloride, sodium and water movement across apical membranes of epithelial cells in the lung, pancreases, and vas deferens. The consequence is dehydration and increased viscosity of mucous secretions leading to obstruction, inflammation and progressive destruction of the affected organs. Activation of other ion channels, particularly chloride channels present in the apical membranes of epithelial cells from CF patients may circumvent the defect in chloride conduction caused by mutations in CFTR. Chloride channels with varying properties have been identified in airway epithelia. We have recently obtained a human cDNA whose predicted amino acid sequence has a high degree of similarity to a voltage-and volume-regulated chloride channel in rat (ClC-2). The human sequence is expressed in a variety of tissues including lung and pancreas and numerous airway epithelial cell lines derived from CF patients. The overall goal of this project is to determine whether the human ClC-2 chloride channel can be manipulated to ameliorate the chloride conduction defect in CF cells by investigation of its functional properties and cellular location. This will be achieved by pursuit of the following aims: 1) To complete the cloning of the 5' end of the hClC-2 cDNA and confirm that it is full-length by determining the start site of transcription. 2) To characterize the chloride currents exhibited by Xenopus oocytes and mammalian 293 cells transiently expressing the full-length hClC-2 cDNA. 3) To determine the cellular location of hClC-2 tagged with a synthetic epitope (FLAG) stably expressed in polarized epithelial cells (MDCK) using apical and basolateral cell membrane markers. 4) To determine which chloride channel is encoded by hClC-2 in epithelial cells by reduction of endogenous hClC-2 using antisense techniques.