Abstract
Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [3H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn2+-binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Aguayo, L.G. et al. Historical and current perspectives of neuroactive compounds derived from Latin America. Mini Rev. Med. Chem. 6, 997–1008 (2006).
Nnadi, C.U., Mimiko, O.A., McCurtis, H.L. & Cadet, J.L. Neuropsychiatric effects of cocaine use disorders. J. Natl. Med. Assoc. 97, 1504–1515 (2005).
Ritz, M.C., Lamb, R.J., Goldberg, S.R. & Kuhar, M.J. Cocaine receptors on dopamine transporters are related to self- administration of cocaine. Science 237, 1219–1223 (1987).
Giros, B., Jaber, M., Jones, S.R., Wightman, R.M. & Caron, M.G. Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606–612 (1996).
Chen, R. et al. Abolished cocaine reward in mice with a cocaine-insensitive dopamine transporter. Proc. Natl. Acad. Sci. USA 103, 9333–9338 (2006).
Torres, G.E. & Amara, S.G. Glutamate and monoamine transporters: new visions of form and function. Curr. Opin. Neurobiol. 17, 304–312 (2007).
Gether, U., Andersen, P.H., Larsson, O.M. & Schousboe, A. Neurotransmitter transporters: molecular function of important drug targets. Trends Pharmacol. Sci. 27, 375–383 (2006).
Iversen, L. Neurotransmitter transporters and their impact on the development of psychopharmacology. Br. J. Pharmacol. 147 Suppl 1: S82–S88 (2006).
Chen, N.H., Reith, M.E. & Quick, M.W. Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch. 447, 519–531 (2004).
Beuming, T., Shi, L., Javitch, J.A. & Weinstein, H. A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na+ symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function. Mol. Pharmacol. 70, 1630–1642 (2006).
Zomot, E. et al. Mechanism of chloride interaction with neurotransmitter:sodium symporters. Nature 449, 726–730 (2007).
Yamashita, A., Singh, S.K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl−-dependent neurotransmitter transporters. Nature 437, 215–223 (2005).
Zhou, Z. et al. LeuT-desipramine structure reveals how antidepressants block neurotransmitter reuptake. Science 317, 1390–1393 (2007).
Singh, S.K., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007).
Uhl, G.R. & Lin, Z. The top 20 dopamine transporter mutants: structure-function relationships and cocaine actions. Eur. J. Pharmacol. 479, 71–82 (2003).
Volz, T.J. & Schenk, J.O. A comprehensive atlas of the topography of functional groups of the dopamine transporter. Synapse 58, 72–94 (2005).
Vaughan, R.A. et al. Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter. J. Biol. Chem. 282, 8915–8925 (2007).
Niv, M.Y. & Weinstein, H. A flexible docking procedure for the exploration of peptide binding selectivity to known structures and homology models of PDZ domains. J. Am. Chem. Soc. 127, 14072–14079 (2005).
Carroll, F.I., Lewin, A.H., Boja, J.W. & Kuhar, M.J. Cocaine receptor: biochemical characterization and structure-activity relationships of cocaine analogues at the dopamine transporter. J. Med. Chem. 35, 969–981 (1992).
Huang, X. & Zhan, C.G. How dopamine transporter interacts with dopamine: insights from molecular modeling and simulation. Biophys. J. 93, 3627–3639 (2007).
Kitayama, S. et al. Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc. Natl. Acad. Sci. USA 89, 7782–7785 (1992).
Ukairo, O.T. et al. Recognition of benztropine by the dopamine transporter (DAT) differs from that of the classical dopamine uptake inhibitors cocaine, methylphenidate, and mazindol as a function of a DAT transmembrane 1 aspartic acid residue. J. Pharmacol. Exp. Ther. 314, 575–583 (2005).
Lin, Z., Wang, W., Kopajtic, T., Revay, R.S. & Uhl, G.R. Dopamine transporter: transmembrane phenylalanine mutations can selectively influence dopamine uptake and cocaine analog recognition. Mol. Pharmacol. 56, 434–447 (1999).
Lee, S.H. et al. Importance of valine at position 152 for the substrate transport and 2β-carbomethoxy-3β-(4-fluorophenyl)tropane binding of dopamine transporter. Mol. Pharmacol. 57, 883–889 (2000).
Henry, L.K. et al. Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high-affinity recognition of antidepressants. J. Biol. Chem. 281, 2012–2023 (2006).
Chen, J.G., Sachpatzidis, A. & Rudnick, G. The third transmembrane domain of the serotonin transporter contains residues associated with substrate and cocaine binding. J. Biol. Chem. 272, 28321–28327 (1997).
Bismuth, Y., Kavanaugh, M.P. & Kanner, B.I. Tyrosine 140 of the gamma-aminobutyric acid transporter GAT-1 plays a critical role in neurotransmitter recognition. J. Biol. Chem. 272, 16096–16102 (1997).
Henry, L.K., Adkins, E.M., Han, Q. & Blakely, R.D. Serotonin and cocaine-sensitive inactivation of human serotonin transporters by methanethiosulfonates targeted to transmembrane Domain I. J. Biol. Chem. 278, 37052–37063 (2003).
Zhou, Y., Bennett, E.R. & Kanner, B.I. The aqueous accessibility in the external half of transmembrane domain I of the GABA transporter GAT-1 Is modulated by its ligands. J. Biol. Chem. 279, 13800–13808 (2004).
Loland, C.J., Norregaard, L. & Gether, U. Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn2+-binding site. J. Biol. Chem. 274, 36928–36934 (1999).
Schwartz, T.W., Frimurer, T.M., Holst, B., Rosenkilde, M.M. & Elling, C.E. Molecular mechanism of 7TM receptor activation—a global toggle switch model. Annu. Rev. Pharmacol. Toxicol. 46, 481–519 (2006).
White, K.J., Kiser, P.D., Nichols, D.E. & Barker, E.L. Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter. Protein Sci. 15, 2411–2422 (2006).
Loland, C.J., Granas, C., Javitch, J.A. & Gether, U. Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding. J. Biol. Chem. 279, 3228–3238 (2004).
Zomot, E., Zhou, Y. & Kanner, B.I. Proximity of transmembrane domains 1 and 3 of the GABA transporter GAT-1 inferred from paired cysteine mutagenesis. J. Biol. Chem. 280, 25512–25516 (2005).
Newman, A.H. & Kulkarni, S. Probes for the dopamine transporter: new leads toward a cocaine-abuse therapeutic—a focus on analogues of benztropine and rimcazole. Med. Res. Rev. 22, 429–464 (2002).
Desai, R.I., Kopajtic, T.A., Koffarnus, M., Newman, A.H. & Katz, J.L. Identification of a dopamine transporter ligand that blocks the stimulant effects of cocaine. J. Neurosci. 25, 1889–1893 (2005).
Loland, C.J. et al. Relationship between conformational changes in the dopamine transporter and cocaine-like subjective effects of uptake inhibitors. Mol. Pharmacol. 73, 813–823 (2008).
Lin, Z. & Uhl, G.R. Dopamine transporter mutants with cocaine resistance and normal dopamine uptake provide targets for cocaine antagonism. Mol. Pharmacol. 61, 885–891 (2002).
Chen, R., Han, D.D. & Gu, H.H. A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate. J. Neurochem. 94, 352–359 (2005).
Sen, N., Shi, L., Beuming, T., Weinstein, H. & Javitch, J.A. A pincer-like configuration of TM2 in the human dopamine transporter is responsible for indirect effects on cocaine binding. Neuropharmacology 49, 780–790 (2005).
Loland, C.J., Norregaard, L., Litman, T. & Gether, U. Generation of an activating Zn2+ switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proc. Natl. Acad. Sci. USA 99, 1683–1688 (2002).
Barker, E.L., Moore, K.R., Rakhshan, F. & Blakely, R.D. Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter. J. Neurosci. 19, 4705–4717 (1999).
Ravna, A.W., Sylte, I. & Dahl, S.G. Molecular model of the neural dopamine transporter. J. Comput. Aided Mol. Des. 17, 367–382 (2003).
Edvardsen, O. & Dahl, S.G. A putative model of the dopamine transporter. Brain Res. Mol. Brain Res. 27, 265–274 (1994).
Katz, J.L., Kopajtic, T.A., Agoston, G.E. & Newman, A.H. Effects of N-substituted analogues of benztropine: diminished cocaine-like effects in dopamine transporter ligands. J. Pharmacol. Exp. Ther. 309, 650–660 (2004).
Brooks, B.R. et al. CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217 (1983).
Bayly, C.I., Ceplak, P.D., C.W., & Kollman, P.A. A well-behaved electrostatic potential–based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269–10280 (1993).
Acknowledgements
We thank P. Elsman and D. Vang Larsen for excellent technical assistance. The work was supported in part by US National Institute of Health Grants P01 DA 12408 (U.G., H.W. and J.A.J.) and DA 22413 (J.A.J.), the Danish Medical Research Council (C.J.L., J.K. and U.G.), the Lundbeck Foundation (C.J.L. and U.G.), the Novo Nordisk Foundation (M.L.B., C.J.L., J.K. and U.G.), the Maersk Foundation (C.J.L.) and the US National Institute on Drug Abuse Intramural Research Program (A.H.N.). J.K. was a recipient of a European Molecular Biology Organization long-term fellowship.
Author information
Authors and Affiliations
Contributions
T.B. designed and performed the computational experiments, analyzed the data and wrote the manuscript draft together with C.J.L. J.K. generated mutants, carried out pharmacological analyses and contributed to the data analysis. M.L.B. and K.R. generated mutants and carried out pharmacological analyses. L.S. contributed to the computational experiments and manuscript refinement. L.G. participated in the design and performance of the computational experiments. A.H.N. contributed with ideas, benztropine analogues and provided expertise in the pharmacology and medicinal chemistry of DAT inhibitors. J.A.J. contributed with ideas and to the design of experiments and writing of the manuscript. H.W. directed the design and performance of the modeling and computational experiments, participated in data analysis and contributed to writing the manuscript. U.G. supervised the project together with C.J.L., designed experiments, analyzed data and wrote the final manuscript. C.J.L. supervised the project together with U.G., designed experiments, generated mutants, performed pharmacological experiments, analyzed data and wrote the manuscript draft together with T.B.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–3, Supplementary Tables 1–4, Supplementary Methods and Supplementary Note (PDF 538 kb)
Rights and permissions
About this article
Cite this article
Beuming, T., Kniazeff, J., Bergmann, M. et al. The binding sites for cocaine and dopamine in the dopamine transporter overlap. Nat Neurosci 11, 780–789 (2008). https://doi.org/10.1038/nn.2146
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nn.2146
This article is cited by
-
Cocaine regulates antiretroviral therapy CNS access through pregnane-x receptor-mediated drug transporter and metabolizing enzyme modulation at the blood brain barrier
Fluids and Barriers of the CNS (2024)
-
Ligand coupling mechanism of the human serotonin transporter differentiates substrates from inhibitors
Nature Communications (2024)
-
Bile acids modulate reinstatement of cocaine conditioned place preference and accumbal dopamine dynamics without compromising appetitive learning
Scientific Reports (2023)
-
The dopamine transporter antiports potassium to increase the uptake of dopamine
Nature Communications (2022)
-
Cocaine-mediated circadian reprogramming in the striatum through dopamine D2R and PPARγ activation
Nature Communications (2020)