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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
1R01DA008379-01A1
Application #
2120859
Study Section
Drug Abuse Biomedical Research Review Committee (DABR)
Project Start
1994-08-15
Project End
1998-06-30
Budget Start
1994-08-15
Budget End
1995-06-30
Support Year
1
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Nyola, Ajeeta; Karpowich, Nathan K; Zhen, Juan et al. (2010) Substrate and drug binding sites in LeuT. Curr Opin Struct Biol 20:415-22
Reith, M E A; Zhen, J; Chen, N (2006) The importance of company: Na+ and Cl- influence substrate interaction with SLC6 transporters and other proteins. Handb Exp Pharmacol :75-93
Zhen, Juan; Chen, Nianhang; Reith, Maarten E A (2005) Differences in interactions with the dopamine transporter as revealed by diminishment of Na(+) gradient and membrane potential: dopamine versus other substrates. Neuropharmacology 49:769-79
Chen, Nianhang; Reith, Maarten E A (2004) Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential. J Neurochem 89:750-65
Chen, Nianhang; Zhen, Juan; Reith, Maarten E A (2004) Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine. J Neurochem 89:853-64
Chen, Nianhang; Rickey, Judy; Berfield, Janet L et al. (2004) Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding. J Biol Chem 279:5508-19
Chen, Nianhang; Rickey, Judy; Reith, Maarten E A (2003) Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations. J Neurochem 86:678-86
Chen, Nianhang; Reith, Maarten E A (2003) Na+ and the substrate permeation pathway in dopamine transporters. Eur J Pharmacol 479:213-21
Wang, Lijuan C; Cui, Xiao-Nan; Chen, Nianhang et al. (2003) Binding of cocaine-like radioligands to the dopamine transporter at 37 degrees C: effect of Na+ and substrates. J Neurosci Methods 131:27-33
Wu, Qun; Reith, Maarten E A; Walker, Q David et al. (2002) Concurrent autoreceptor-mediated control of dopamine release and uptake during neurotransmission: an in vivo voltammetric study. J Neurosci 22:6272-81

Showing the most recent 10 out of 33 publications