This proposal describes studies designed to understand the mechanisms by which drugs such as cocaine and neurotoxic amphetamine derivatives interact with proteins that mediate biogenic amine transport. The experiments focus on the serotonin, norepinephrine, and dopamine transport systems responsible for biogenic amine influx across the plasma membrane and the biogenic amine transporter that catalyzes transport into secretory granules. These transporters are responsible for the neuronal reuptake and storage of serotonin, dopamine, and norepinephrine which terminates these transmitters' action at the synapse and recycles transmitter to the synaptic vesicle. Transport studies will assess the ability of neurotoxic amphetamine derivatives to serve as substrates or inhibitors of the serotonin transporter. Binding studies with membranes containing a single monoamine transport system will provide information about the interaction between the cocaine and monoamine binding sites. The ionic form of the substrate amino group in biogenic amine transporters will be investigated using novel fluorinated substrate analogues. cDNA libraries will be screened with a cDNA clone for the norepinephrine transporter. In parallel, plasmid cDNA libraries will be screened by transfection into cultured mammalian cells for expression cloning of the serotonin transporter. Cloned transporter cDNA will be used to search for other serotonin transporters and also for the dopamine transporter by nucleic acid homology. Site-directed polyclonal antibodies will be used to elucidate the transporter tertiary structure. Expression of the cloned transporter in transfected mammalian cells will allow better understanding of the biosynthesis, stoichiometry, electrogenicity and mechanism of the plasma membrane biogenic amine transporters. Such expression systems will be used to probe the relationship between structure and function in membrane transport and its inhibition by cocaine and other drugs.
Showing the most recent 10 out of 14 publications
