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.
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