The binding sites for benztropines and dopamine in the dopamine transporter overlap
Introduction
The dopamine transporter (DAT) mediates rapid reuptake of dopamine from the synaptic cleft and is thereby responsible for termination of dopaminergic signalling (Chen et al., 2004, Gether et al., 2006, Torres and Amara, 2007). Alteration in dopamine signalling and DAT function is coupled to neurological and psychiatric diseases including schizophrenia, bipolar disorder, ADHD (Attention Deficit Hyperactivity Disorder), Tourette’s syndrome and Parkinson’s disease (Gainetdinov and Caron, 2003, Gether et al., 2006, Torres and Amara, 2007). The transporter belongs to the family of neurotransmitter:sodium symporters (NSS) (also called the SLC6 [solute carrier 6] family or Na+/Cl− coupled transporters) that in addition includes the transporters for other neurotransmitters such as the norepinephrine transporter (NET), the serotonin transporter (SERT), the glycine transporters and the GABA transporters (Chen et al., 2004, Gether et al., 2006, Torres and Amara, 2007). NSS proteins utilize the transmembrane Na+ gradient as a driving force for transport of substrate and are characterized by additional co-transport of Cl− (Chen et al., 2004, Gether et al., 2006, Torres and Amara, 2007, Zomot et al., 2007). Insight into the structural basis of NSS function has emerged from crystallographic analysis of LeuT, a bacterial NSS homolog from Aquifex aeolicus (Krishnamurthy et al., 2009, Singh et al., 2008, Singh et al., 2007, Yamashita et al., 2005).
DAT is the principal target for psychostimulants including cocaine and amphetamines (Chen et al., 2004, Gether et al., 2006, Torres and Amara, 2007). Because of wide abuse of these compounds it has been a long pursued goal to develop medication that can aid in the treatment of this addiction, but success has been elusive. In this pursuit, novel dopamine uptake inhibitors have been targeted as leads to potential medications for cocaine addiction (Dutta et al., 2003). Among these compounds are analogs of benztropine (BZT) and rimcazole that have similar or higher affinity and selectivity for the DAT than cocaine (Newman and Katz, 2009, Newman and Kulkarni, 2002). The BZTs tested so far readily cross the blood–brain barrier (Raje et al., 2003, Syed et al., 2008) and produce increases in extracellular levels of dopamine even for longer durations than cocaine (Raje et al., 2005). Nonetheless, several of these DAT inhibitors are less effective than cocaine as behavioral stimulants (Desai et al., 2005, Katz et al., 2004, Katz et al., 2003, Newman et al., 1994). Furthermore, one BZT analog, JHW007, was found to potently antagonize the behavioral effects of cocaine (Desai et al., 2005, Hiranita et al., 2009).
We have demonstrated recently a remarkable relationship between cocaine-like subjective effects of uptake inhibitors and the conformational state promoted by these compounds in the DAT protein (Loland et al., 2008). Our data suggested that the stimulatory effect of an inhibitor might be predicted directly from its interaction mode with the transporter (Loland et al., 2008). Whereas cocaine and its analogs induced an outside-open conformation of the transporter, several BZTs with reduced stimulatory effect, including BZT itself and the analog JHW007, promoted a less outward-open configuration of the transport protein (Loland et al., 2008). Subsequent molecular models of the binding sites in DAT, based on the LeuT structure and a previously reported sequence alignment (Beuming et al., 2006), suggested that the data were consistent with binding sites for both cocaine and the BZTs being deeply buried in the transporter structure and overlapping with that of dopamine (Beuming et al., 2008). Moreover, the models suggested that whereas cocaine and the cocaine analog CFT ((−)-2β-carbomethoxy-3β-(4-fluorophenyl)tropane) stabilized a rather open conformation of the binding pocket by breaking a stabilizing intramolecular hydrogen bond between Asp791.45 and Tyr1563.50, BZT and JHW007 bound in the model with a more closed configuration in which the hydrogen bond between the two residues was not broken (Beuming et al., 2008). In agreement with this binding model, mutational disruption of the hydrogen bond by mutating Tyr1563.50 to Phe did not affect binding of cocaine and CFT but decreased the binding affinity of BZT and JHW007, indicating an indirect role of the hydrogen bond in stabilizing the BZT/JHW007 binding mode (Beuming et al., 2008).
The deeply buried binding site for cocaine and analogs was further validated with detailed mutational analysis (Beuming et al., 2008). However, a similar detailed analysis validating the proposed binding site for BZTs was not done, and the characteristics of the binding site were not thoroughly explored. The specific interest in the mode of binding and the position of the binding site within the transporter for BZTs was rekindled by the results of a recent analysis of the binding site for antidepressants and small molecule inhibitors of the closely related serotonin transporter (SERT). Thus, the crystallographic analysis of low affinity antidepressant binding to LeuT, together with mutational analysis in SERT, has been interpreted as evidence for the binding of SERT inhibitors, including tricyclic antidepressants and selected SSRIs (selective serotonin reuptake inhibitors) in a vestibule extracellular to this pocket (Singh et al., 2007, Zhou et al., 2007, Zhou et al., 2009) rather than deep in the transporter structure, in the primary substrate-binding pocket. Notably, the vestibule had been proposed to harbor a secondary substrate-binding site (S2) in LeuT with a critical role in the translocation mechanism (Shi et al., 2008, Zhao et al., 2010). We provide here strong evidence that BZT and BZT analogs can bind to the primary substrate-binding pocket and thus that the compounds display a classical competitive binding mode with binding sites overlapping that of cocaine and dopamine.
Section snippets
Molecular biology and cell culture
Synthetic cDNAs encoding the human DAT (synDAT) were subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) (Loland et al., 2004). All mutations were generated by the QuickChange method (adapted from Stratagene, La Jolla, CA) and confirmed by restriction enzyme mapping and DNA sequencing. COS7 cells were grown and transiently transfected by use of Lipofectamine™ 2000 Transfection Reagent according to the instructions from the manufacturer (Invitrogen).
Indexing of residues
A generic numbering scheme for amino acid
Results
Docking models for the binding of dopamine, cocaine, and selected inhibitors including CFT, BZT and the BZT analog JHW007 had been described previously (Beuming et al., 2008). We based these models on the high-resolution structure of the bacterial NSS member, LeuT (PDB code 2A65) (Yamashita et al., 2005), and a refined structure-based sequence alignment of NSS proteins (Beuming et al., 2006). The initial docked complexes were further refined with molecular dynamics (MD) simulations in explicit
Discussion
In a recent study we provided evidence that the binding site in DAT for cocaine and cocaine analogs overlap with the binding site of the substrate dopamine (Beuming et al., 2008). Thus, we obtained data arguing strongly against an allosteric mechanism of dopamine binding inhibition for cocaine (Beuming et al., 2008). Our previous dockings also proposed a binding site for BZT and BZT analogs in the primary substrate-binding cavity (S1) of DAT and, hence, overlapping binding sites with dopamine
Acknowledgments
We thank Pia Elsman for technical assistance and Jianjing Cao for synthesizing the BZT analogs used in this study. The work was supported in part by the National Institute of Health Grants P01 DA12408 (HW and UG), R00 DA023694 (LS), the NIDA-Intramural Research Program (A.H.N), the Danish Health Science Research Council (CJL and UG), the Lundbeck Foundation (CJL and UG), the Novo Nordisk Foundation (CJL and UG), and the Maersk Foundation (CJL).
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These authors contributed equally to this work.
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Number in superscript describes residue number according to generic numbering scheme (see Materials and Methods).