MOLECULAR BIOLOGY OF CL CHANNEL IN CYSTIC FIBROSIS

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Cystic fibrosis is now known to be due to a recessive genetic defect in the regulation of anion transport in epithelial cells. In these epithelia, chloride is transported across the cell membrane through a channel that can be opened by cyclic AMP dependent protein kinase. Recently, it was shown that in cystic fibrosis, the kinase fails to open the channel, even though the channel is present in the membrane. The Columbia-Presbyterian Cystic Fibrosis Center is composed of a multidisciplinary group of investigators who are collaborating towards identification of the molecular defect in this disease. We have already purified the epithelial chloride channel and using this procedure we will clone and sequence the gene for the channel. Methods have been developed for expression of ion transport proteins in frog oocytes which will facilitate the cloning of the chloride channel. Another expression system was developed which relies on DNA-mediated gene transfer, that will allow the cloning of iodide and chloride transport proteins from thyroid epithelial cells. This method will also be used to identify structural and regulatory mutants of these genes. Regulation of the chloride channel by a variety of protein kineses will be studied in human epithelial cells and in cells derived from patients with cystic fibrosis. The chloride channel will be reconstituted in planar lipid bilayer and the effect of kineses on its electrophysiological characteristics will be studied. The Center will promote the collaboration of all the involved groups towards the identification of the genetic defect in cystic fibrosis.