New information obtained during the past five years has pointed to an abnormality in secretory chloride channel regulation as the basic defect in cystic fibrosis (CF). The apical membrane-located chloride channel represents the rate-limiting step for Cl- secretion and, thus, influences the composition of the secretory products of the airways, pancreas, and sweat glands, the major target organs in CF. Although much important knowledge has been learned about the phenomenology of epithelial secretory Cl- channel kinetics and regulation by cAMP-and Ca++-dependent pathways, virtually nothing is known about the structural details of the protein(s) comprising the anion channel complex. The biochemical identification of secretory Cl- channels has been hampered by the lack of suitable probe molecules. The central goal of this Cystic Fibrosis Research Center application is to isolate and characterize this secretory C1- channel protein complex and any associated regulatory components so that a molecular basis for CF can be specified. The development of this proposal revolves around the generation of antibodies that appear to interact specifically with the secretory Cl- channel-containing vesicles. Four interdisciplinary, interrelated projects are proposed: 1) isolation of secretory Cl- channels using antibody probes: 2) cloning and expression of the secretory Cl- channel; 3) immunocytochemical localization and intracellular trafficking of Cl- channels; and 4) disulfonic stilbenes as probes of reconstituted Cl- channels. Thus, the secretory Cl- channel will be studied at both the molecular and physiological levels to [provide a comprehensive framework for understanding the mechanisms of anion conduction and regulation. By concentrating our efforts on the molecular and cell biological aspects of C1- channel structure and movement, new information will be generated that will undoubtedly lead to novel strategies for pharmacological intervention to overcome the regulatory deficiencies occurring in CF.
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