In the previous stage of this project, supported by DA 08379-01 through 03 we have characterized cocaine binding sites on the dopamine transporter (DAT) in relation to sites for other uptake blockers, DA, and ions, as well as their regulation by protein kinase C and arachidonic acid. Studies included both the rat DAT in situ and the human DAT expressed by host cell systems such as C6 glioma and HEK 293 cells. Evidence for DAT dimers under non-reducing conditions was obtained in experiments in which photoaffinity-labeled rat DAT was immunoprecipitated with anti-DAT antibody. Cysteine mutants of the human DAT were prepared by site-directed mutagenesis. In the present application, we propose to further study the relationships between cocaine binding and the substrate recognition domains on the DAT including the role of cysteine residues. Human DAT cysteine mutants will be prepared and tested for DAT cell surface expression (biotinylation), DAT function (DA uptake and blocker binding), sensitivity towards sulfhydryl reagents (membrane permeable and impermeable), and their capability to form dimers (co-transfection of wild-type and mutant hDAT cDNA). The impact of palmitoylation of cysteine residues on DAT function will be studied, both in the human DAT expressed in C6 glioma cells and the in situ rat DAT. The interaction of Na+, K+, C1- and H+ with hDAT function will be assessed in preparations of membranes or plasma membrane vesicles of cells expressing the human DAT. The latter system will allow studying the impact of ion gradients and membrane potential on DAT function as well as determining which form of DA, neutral or protonated, is translocated. Finally, the spare receptor concept will be tested for the DAT by classical pharmacological approaches involving irreversible blockade by the cocaine analog RTI-76 as well as molecular techniques addressing oligomerization, both for the human DAT in a host system and the rat DAT in situ.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
7R01DA008379-10
Application #
6801757
Study Section
Special Emphasis Panel (ZDA1-GXD-C (07))
Program Officer
Lin, Geraline
Project Start
1994-08-15
Project End
2005-08-31
Budget Start
2003-10-01
Budget End
2005-08-31
Support Year
10
Fiscal Year
2003
Total Cost
$177,934
Indirect Cost
Name
New York University
Department
Psychiatry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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; 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
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
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

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