This Physician Scientist Award application is based in the Departments of Physiology and Pediatrics at Tufts University School of Medicine, and The Boston Floating Hospital for Infants and Children, New England Medical Center Hospitals. The applicant has assembled a group of senior scientists to supervise Phase I and to guide Phase II of the research plan, and appropriate commitments have been made by the Departments of Pediatrics for a future faculty position for the applicant in the fourth year of the program. The long range goals of the research described are to elucidate the structure and function of the coated vesicle chloride channel. This Cl- channel is essential for the acidification of vacuolar compartments by ATP-driven proton pumps present in endosomes, Golgi- derived vesicles and lysosomes. Cl- conductance provides the compensating charge balance to electrogenic proton transport. Acidification is intimately involved in the regulation of various cellular processes, including receptor-mediated endocytosis, ligand dissociation and membrane trafficking within the cell.
The specific aims of Phase I of the proposal seek: 1) to validate Cl- conductance as measured through SPQ fluorescence quenching and potential-driven 36Cl flux in clathrin coated and reconstituted vesicles; 2) to purify the Cl- channel from coated vesicles using monoclonal antibodies and density shift centrifugation; 3) to characterize the regulation, if any, of this Cl- channel by modulators including phosphorylation, Ca2+, eicosanoids, and G proteins; 4) to characterize its electrophysiological properties by patch clamping; and 5) to elucidate the similarities and differences of coated vesicle Cl- channels and those in epithelial membranes. In Phase II, studies will be devoted to understanding secretory processes in immortalized bile duct epithelial cells obtained from patients with cystic fibrosis and normal controls. This part of the program will rely on techniques validated in Phase I of the project. Chloride permeability plays a role in a number of cellular functions in various intracellular and epithelial membranes. The relationship of epithelial to intracellular Cl- channels remains to be elucidated, and is the focus of this research plan for the next 5 years. These studies should improve understanding of the regulation of intracellular chloride channels, will begin to identify secretory processes of biliary epithelium, and should also determine whether the defective CAMP-dependent phosphorylation of the epithelial Cl- channel in cystic fibrosis is expressed in intracellular chloride channels.
