The ability of cocaine to inhibit dopamine reuptake in areas of the brain such as the nucleus accumbens where dopamine is reinforcing, is believed to be a major mechanism contributing to the abuse potential of cocaine. Although we and others have characterized the binding of cocaine and other tritiated dopamine reuptake inhibitors to the dopamine reuptake transporter protein less is known about the molecular mechanism by which the binding of a ligand such as cocaine to a sodium dependent transporter results in inhibition of reuptake. Recently, we synthesized a tritiated azido diphenylpiperazine derivative 1-[2bis(phenyl)methoxy]ethyl- 4-[3-(3'-azidophenyl)-{2,3-3H]propyl}piperazine (3azido[3H]GBR-12935.), which can be covalently incorporated (in a sodium dependent manner and with appropriate pharmacological specificity) into a single striatal 80 kDa polypeptide of the dopamine reuptake transporter protein (as detected by sodium dodecyl sulfate-polyacrylamine gel electrophoresis and fluorography). This grant proposes a series of studies that will attempt to determine the molecular composition of the dopamine transporter as well as identify regions of the transporter protein to which uptake inhibitors bind. Specific questions to be addressed are whether there are different regional isoforms of the dopamine transporter, whether several dopamine reuptake inhibitor share, a common molecular mechanism and whether dopamine reuptake inhibitors interact with the substrate (dopamine) binding site on the transporter. Methodologies proposed include further covalent incorporation studies with 3-azido [3H]GBR-12935 and comparison to covalent incorporation studies of other pharmacological probes (BTCP, metaphit, cocaine isothiocyanate, xylamine). Radiation inactivation studies are proposed to determine whether cocaine and other dopamine reuptake inhibitors interact with a protein of similar molecular weight (preliminary data suggests this is not the case). Finally chemical modification / inactivation studies are proposed to identify, compare and contrast important functional groups for the binding of cocaine and other dopamine reuptake inhibitors.
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