Chloride channels are present in epithelia where they mediate salt absorption and secretion. In many epithelial cells, chloride channels are activated by cyclic AMP, a regulatory process that is deranged in cystic fibrosis and cholera. To purify the chloride channel requires the use of a ligand that can bind to the channel with a high affinity so that one can follow the channel during its purification. We developed a number of inhibitory ligands and characterized the binding sites for their tritiated derivatives in channel-containing membrane vesicles isolated from kidney and tracheal mucosa. We also synthesized an affinity matrix based on the structure of one of these inhibitors. After solubilizing the binding site we were able to reconstitute it into liposomes which transported 36C1, aflux that was inhibitable by the high affinity drug. We started the purification of the channel using ligand affinity and other standard chromatographic procedures. We will reconstitute the purified channel protein into liposomes and characterize its behavior. We will develop antibodies to the purified channel and use them for localization of the channel in different epithelia. We will study phosphorylation of the channel in purified reconstituted vesicles using defined kinases such as the cyclic AMP dependent protein kinase. We will prepare peptides from the purified protein, sequence them and prepare oligonucleotide probes. These and the antibodies will be used to clone the gene for the chloride channel from a tracheal cell cDNA library. These studies will allow us to define the molecular defect in cystic fibrosis, the commonest lethal genetic disease in the white population of this country.
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