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 [ 3 H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn 2+ -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.Correspondence should be addressed to U.G. (E-mail: gether@sund.ku.dk). Note: Supplementary information is available on the Nature Neuroscience website. AUTHOR CONTRIBUTIONST.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.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2009 July 1. Published in final edited form as:Nat Neurosci. 2008 July ; 11(7): 780-789. doi:10.1038/nn.2146. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCocaine is an alkaloid derived from the Peruvian Erythroxylon coca plant and has been used as a stimulant for centuries 1 . Today, cocaine is widely abused, especially in the western hemisphere, causing major socioeconomic burdens through increased medical expenses, lost earnings and increased crime 2 . Nonetheless, the molecular mechanisms underlying cocaine's pharmacology and abuse ...

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

“…However, an apparent paradox exists in theory: It is understandable that decreasing MAOB after cocaine treatment should enhance the acute action of cocaine, and yet, on the contrary, deprenyl blocked the action of cocaine during withdrawal. We can only speculate at present that deprenyl, given before cocaine during withdrawal, should have raised up the localized basal level of DA that is known to compete with cocaine at DAT as a competitive inhibitor (52,53), thus attenuating the action of cocaine. Further study is certainly warranted in this regard.…”

Transmembrane Domain 6 of the Human Serotonin Transporter Contributes to an Aqueously Accessible Binding Pocket for Serotonin and the Psychostimulant 3,4-Methylene Dioxymethamphetamine

“…Although the data suggest protection at S336C, this effect was small and did not reach significance. In addition, hDAT Phe-326 and Val-328, which correspond by homology to hSERT Phe-341 and Val-343, are predicted to have side chains that interact with the tropane ring of the cocaine-like molecule CFT (16). These findings suggest that, in hSERT, cocaine may orient in a conformation distinct from that adopted in hDAT.…”

“…Modeling and mutagenesis studies of cocaine and cocaine analog binding in hDAT predict that cocaine would interact significantly with hSERT TM6 residues (16,57,58). Specifically, hDAT residues Phe-320, Ser-321, and Leu-322, which correspond by homology to hSERT Phe-335, Ser-336, and Leu-337, are predicted to interact with the N-methylamine of (Ϫ)-2␤-carbomethoxy-3␤-(4-fluorophenyl)tropane (CFT), a cocaine analog, and thus, we predicted that the homologous hSERT residues would be protected by cocaine.…”

Binding of the Amphetamine-like 1-Phenyl-piperazine to Monoamine Transporters

“…Especially, the mechanisms by which the so-called monoamine releasers, for example, MDMA, phenyl-piperazine (PP), and some PP analogs as 1-(3-chlorophenyl)-piperazine (mCPP) and 1-(3-trifluoromethylphenyl)-piperazine (TFMPP or "Legal X") reverse the direction of the normal transport pathway of 5-HT, thereby causing efflux, are not well understood. While the inhibitor cocaine, which is similar to certain antidepressants, 9 relies on endogenous tone by inhibiting the uptake of already released neurotransmitter by the monoamine transporters, the releasers are substrates of the monoamine transporters. The releasers thereby both compete for the uptake site with neurotransmitter and also cause efflux of the neurotransmitter through the transporter.…”