Biogenic amine transporters are responsible for terminating the synaptic action of serotonin (5-HT), dopamine (DA), and norepinephrine (NE). They are the molecular targets for antidepressants such as imipramine and Prozac as well as for psychostimulants such as cocaine and amphetamines. Efforts currently directed at designing cocaine antagonists make assumptions about the proximity of cocaine and biogenic amine binding sites on the transporter. This application describes experiments designed to map the location of amino acid residues in biogenic amine transporters that are involved with substrate and inhibitor binding. These experiments will provide evidence to test the assumptions now being used to design cocaine treatment medications. Biogenic amine transporters use transmembrane ion gradients to drive neurotransmitter uptake. The coupling stoichiometry for this process is characteristic of each transporter. This proposal outlines plans to determine the ion coupling stoichiometry for DA transport and to compare it with the stoichiometry for NE and 5-HT transport. Mutant transporters will be tested to determine how the stoichiometry of an individual transporter depends on individual amino acids. Taken together, the experiments outlined in this proposal are directed toward the ultimate goal of understanding how each part of the primary sequence of a biogenic amine transport protein participates in the binding and the translocation of substrates.
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