INVESTIGATION OF ALTERNATE CHLORIDE CHANNELS

囊性纤维化（CF）是最常见的致死性常染色体隐性遗传病之一， 高加索人的疾病 它是由CF突变引起的。 跨膜电导调节器（CFTR）。 这种蛋白质的功能是 功能性CFTR导致氯、钠和水运动异常 穿过肺、胰腺和胰腺中上皮细胞的顶膜， 输精管 其结果是脱水和粘度增加， 粘液分泌物导致阻塞、炎症和进行性 破坏受影响的器官。 激活其他离子通道， 特别是存在于顶膜中的氯离子通道， 来自CF患者的上皮细胞可以绕过氯离子缺陷， CFTR突变引起的传导。 氯离子通道 已经在气道上皮中鉴定了这些特性。 我们最近 获得了人cDNA，其预测的氨基酸序列具有高的 与电压和容量调节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 包括肺和胰腺以及许多气道上皮细胞系 来自CF患者。 该项目的总体目标是 确定是否可以操纵人ClC-2氯离子通道， 改善CF电池的氯离子传导缺陷 它的功能特性和细胞位置。 这将是 通过追求以下目标实现： 1)为了完成hClC-2 cDNA 5'端的克隆， 通过确定转录的起始位点来确定它是全长的。 2)为了表征爪蟾卵母细胞的氯电流， 瞬时表达全长hClC-2 cDNA的哺乳动物293细胞。 3)为了确定用合成的标记物标记的hClC-2的细胞定位， 在极化上皮细胞（MDCK）中稳定表达的表位（FLAG） 使用顶侧和基底侧细胞膜标记。 4)为了确定上皮细胞中hClC-2编码哪个氯通道， 细胞通过使用反义技术减少内源性hClC-2。

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.