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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 ...

The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family display favorable behavior relative to conventional detergents, as tested on multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied.

Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine-and rimcazolebased inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conformation, whereas benztropine and rimcazole analogs bind a closed conformation. Next, we investigated the changes in inhibition potency of [ 3 H]dopamine uptake of the compounds at a mutant DAT (Y335A) characterized by a global change in the conformational equilibrium. We observed a close relationship between the decrease in potencies of inhibitors at this mutant and cocaine-like responding in rats trained to discriminate cocaine from saline injections. Our data suggest that chemically different DAT inhibitors stabilize distinct transporter conformations and that this in turn affects the cocaine-like subjective effects of these compounds in vivo.

Background (±)-Modafinil has piqued interest as a treatment for ADHD and stimulant dependence. The R-enantiomer of modafinil may have unique pharmacological properties that should be further investigated. Methods (±)-Modafinil and its R-(−)- and S-(+)-enantiomers were synthesized and tested for inhibition of [3H]DA uptake and [3H]WIN 35,428 binding in hDAT WT and mutants with altered conformational equilibria. Data were compared to cocaine and the atypical dopamine uptake inhibitor, JHW 007. R- and S-modafinil were also evaluated in microdialysis studies in the mouse NAc shell and in a cocaine discrimination procedure. Results (±)-, R- and S-Modafinil bind to the DAT and inhibit dopamine uptake less potently than cocaine, with R-modafinil having ~3-fold higher affinity than its S-enantiomer. Molecular docking studies revealed subtle differences in binding modes for the enantiomers. R-modafinil was significantly less potent in the DAT Y156F mutant compared to wild-type DAT, whereas S-modafinil was affected less. Studies with the Y335A DAT mutant showed that the R- and S-enantiomers tolerated the inward facing conformation better than cocaine, which was further supported by MTSET reactivity on the DAT E2C I159C. Microdialysis studies demonstrated that both R- and S-modafinil produced increases in extracellular DA concentrations in the NAc shell less efficaciously than cocaine, and with a longer duration of action. Both enantiomers fully substituted in mice trained to discriminate cocaine from saline. Conclusions R-modafinil displays an in vitro profile different from cocaine. Future trials with R-modafinil as a substitute therapy with the potential benefit of cognitive enhancement for psychostimulant addiction are warranted.

Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we analyzed a cohort of patients with atypical movement disorder and identified 2 DAT coding variants, DAT-Ile312Phe and a presumed de novo mutant DAT-Asp421Asn, in an adult male with early-onset parkinsonism and ADHD. According to DAT single-photon emission computed tomography (DAT-SPECT) scans and a fluoro-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine, and electrophysiological analyses identified a large cation leak that might further perturb dopaminergic neurotransmission. Our results link specific DAT missense mutations to neurodegenerative early-onset parkinsonism. Moreover, the neuropsychiatric comorbidity provides additional support for the idea that DAT missense mutations are an ADHD risk factor and suggests that complex DAT genotype and phenotype correlations contribute to different dopaminergic pathologies.

Binding of Zn 2؉ to the endogenous Zn 2؉ binding site in the human dopamine transporter leads to potent inhibition of [ 3 H]dopamine uptake. Here we show that mutation of an intracellular tyrosine to alanine (Y335A) converts this inhibitory Zn 2؉ switch into an activating Zn 2؉ switch, allowing Zn 2؉ -dependent activation of the transporter. The tyrosine is part of a conserved YXX⌽ trafficking motif (X is any residue and ⌽ is a residue with a bulky hydrophobic group), but Y335A did not show alterations in surface targeting or protein kinase C-mediated internalization. Despite wild-type levels of surface expression, Y335A displayed a dramatic decrease in [ 3 H]dopamine uptake velocity (Vmax) to less than 1% of the wild type. In addition, Y335A showed up to 150-fold decreases in the apparent affinity for cocaine, mazindol, and related inhibitors whereas the apparent affinity for several substrates was increased. However, the presence of Zn 2؉ in micromolar concentrations increased the V max up to 24-fold and partially restored the apparent affinities. The capability of Zn 2؉ to restore transport is consistent with a reversible, constitutive shift in the distribution of conformational states in the transport cycle upon mutation of Tyr-335. We propose that this shift is caused by disruption of intramolecular interactions important for stabilizing the transporter in a conformation in which extracellular substrate can bind and initiate transport, and accordingly that Tyr-335 is critical for regulating isomerization between discrete states in the transport cycle.

Neurotransmitter:sodium symporters (NSS)1 mediate sodiumdependent reuptake of neurotransmitters from the synaptic cleft and are targets for many psychoactive drugs. The crystal structure of the prokaryotic NSS protein, LeuT, was recently solved at high resolution; however, the mechanistic details of regulation of the permeation pathway in this class of proteins remain unknown. Here we combine computational modeling and experimental probing in the dopamine transporter (DAT) to demonstrate the functional importance of a conserved intracellular interaction network. Our data suggest that a salt bridge between Arg-60 in the N terminus close to the cytoplasmic end of transmembrane segment (TM) 1 and Asp-436 at the cytoplasmic end of TM8 is stabilized by a cation-interaction between Arg-60 and Tyr-335 at the cytoplasmic end of TM6. Computational probing illustrates how the interactions may determine the flexibility of the permeation pathway, and mutagenesis within the network and results from assays of transport, as well as the state-dependent accessibility of a substituted cysteine in TM3, support the role of this network in regulating access between the substrate binding site and the intracellular milieu. The mechanism that emerges from these findings may be unique to the NSS family, where the local disruption of ionic interactions modulates the transition of the transporter between the outward-and inward-facing conformations.