In the original application it was noted that there was increasing evidence suggesting that alterations in anion transport were responsible for many, if not all, the epithelial abnormalities in patients with Cystic Fibrosis (CF). The original goals were to characterize the abnormal transport, the presumed defective chloride transport protein, and ultimately to determine the DNA sequence associated with the protein. Early in the course of study of epithelial chloride fluxes it became evident that a system for the continuous monitoring of chloride efflux was essential because of the very rapid nature of the phenomenon. We have developed a system to measure chloride fluxes from cells and reconstituted vesicles and capable of responding to fluxes with T(1/2)s less than one second. We are using the methodology to monitor purification of the chloride channel believed to be associated with the CF defect.
The aim of the continuation is to complete purification of the chloride channel associated with CF. It is expected that considerable homology should exist between the channel of the CF airway and those responsible for conductive chloride in the airways of other mammalian species. For ease in obtaining large amounts of tracheal epithelial tissue from which to purify the conductive chloride channel, we have chosen to use the bovine trachea as our source. The bovine tracheal chloride channels we have been studying have no suitable irreversible inhibitors of chloride transport that might allow identification of the channel on an SDS gel. As a result, our approach to purification makes use of the method of transport specific fractionation. After isolation, the purified channel proteins will be used to produce antibodies. They will also be used to obtain partial sequences of the channel protein. The antibodies and/or partial protein sequence information will be used to clone the gene(s) for the channel. The channel distribution in various tissues will be examined using antibodies and the cloned cDNAs. A long-term objective of this work is to isolate the gene for the chloride channel from humans, and to determine whether the structure of the channel is altered in individuals with CF.
