The potent blockade of the neuronal uptake of dopamine by cocaine is thought to be important for its rewarding effects. Although a great deal of information has been obtained regarding the inhibition of dopamine uptake by cocaine, the nature of the interaction between cocaine and the dopamine carrier is still under debate. This proposal has the following specific aims: 1. To further characterize cocaine binding sites - Cocaine binding sites will be labeled with [3H]CFT and [3H]CIT and the binding characteristics will be compared with those of other radioligands targeted at the dopamine transporter. Different properties would make it likely that different binding domains are involved. 2. To examine regional differences in the dopamine transporter - The hypothesis will be tested that its properties are different in terminal and somatodendritic regions with the high-affinity probe[125I]RTI-121. 3. To investigate the relationship between sites for cocaine and sites for other blockers of dopamine uptake. The question of whether these sites are separate, identical, or otherwise interact (allosterically?) will be addressed through equilibrium and kinetic binding studies with various radioligands. The interactions between blockers binding to multiple sites could be very different from those of blockers binding to one and the same site. The possible existence of partial agonists among dopamine uptake blockers will be addressed in microdialysis experiments in awake animals and in studies with superfused brain slices. 4. To assess the relationships between cocaine sites and the substrate recognition site for dopamine uptake - Similar questions as above will be addressed for these sites in equilibrium/kinetic binding studies and in translocation experiments with synaptosomes and plasma membrane vesicles. Protection by blockers and substrates will be studied against inactivation by acylators and sulfhydryl reagents. The existence of cocaine sites separate from the substrate recognition site would make the existence of a cocaine antagonist, itself devoid of dopamine uptake blocking activity, possible. 5. To investigate the regulation of the dopamine transporter by phosphorylation mechanisms and its sensitivity to cocaine - Extracellular or intracellular phosphorylation mechanisms will be studied by examining the effect of addition of protein kinase C, antagonizing endogenous ATP action, raising intracellular cAMP, or activating/inhibiting endogenous protein kinases.
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