This project will study the structure and function of monoamine transporters. Our approach builds upon cDNA cloning and heterologous expression in Xenopus oocytes. Functional measurements will include flux of radiolabeled substrates, binding of ligands, electrophysiology, and electrochemical detection of monoamines. 1. Cloning and characterization of additional monoamine transporters will continue. Several partial cDNA clones from Drosophila will be used to screen for full-length clones, these will be sequenced and characterized functionally. Present and future Drosophila clones will be used as probes to isolate possible homologous mammalian transporters. 2. Physiological studies will continue on cloned monoamine transporters in heterologous expression systems. Dopamine and norepinephrine transporters will be characterized with methods proven for 5-HT transporters. The discrepancy between integrated current flow and [3H]5-HT uptake for 5-HT transporters will be studied. The nature of the irreversible transition to high leakage current levels that occurs with a mammalian 5-HT transporter will be studied. 3. Structure-function analysis will be performed with monoamine transporters. Experiments will utilize chimeras and site-directed mutations. The focus will be on localizing those domains and residues responsible for pharmacological properties and for permeation properties. 4. Systems will be developed for high-level expression with the eventual goal of structural studies. Expression will be optimized using the available techniques and assays. Initial characterization will utilize freeze-fracture and atomic force microscopy. Monoamine transporters are the targets for (a) many drugs of abuse, (b) many antidepressants, and (c) a toxin that produces Parkinsonism.
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