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The binding sites for cocaine and dopamine in the dopamine transporter overlap

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.

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Figure 1: Models of DAT/ligand complexes.
Figure 2: Evidence for involvement of Val152, Tyr156, Asn157, Val328 and Ser422 in binding of dopamine and/or CFT by DAT.
Figure 3: Validation of the CFT docking model using intramolecular cross-linking and cysteine-reactive reagents.
Figure 4: Molecular docking models of cocaine, amphetamine (AMPH) and JHW007 with experimental validation.
Figure 5: Comparison of the CFT-binding mode with the binding mode of dopamine and other DAT ligands.

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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.

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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.

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Correspondence to Ulrik Gether.

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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

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