The binding sites for cocaine and dopamine in the dopamine transporter are overlapping

Thijs Beuming(a), Marianne L. Bergmann(b), Lei Shi(c), Luis Gracia(a), Klaudia Raniszewska(b), Amy Hauck Newman(e), Jonathan A. Javitch(d), Harel Weinstein(a,c), Ulrik Gether(b), and Claus J. Loland(b)

1. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021
2. Molecular Neuropharmacology Group, Department of Neuroscience and Pharmacology, The Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark
3. Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, NY 10021
4. Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032
5. Medicinal Chemistry Section, Medications Discovery Research Branch, National Institute on Drug Abuse, Intramural Research Program, Department of Health and Human Services, National Institutes of Health, Baltimore, Maryland 21224, USA

Cocaine is a widely abused substance with psychostimulant effects attributed to inhibition of the presynaptic dopamine transporter (DAT) a member of the neurotransmitter:sodium symporter (NSS) family; however, the molecular mechanism underlying this inhibition has remained largely unknown. Here, we present the first molecular models for DAT binding of cocaine and cocaine-analogues based on the high-resolution structure of the bacterial transporter homologue, LeuT5. Our models, which were validated by detailed mutagenesis, suggest that the binding site for cocaine and cocaine-analogues is deeply buried between transmembrane segments (TM) 1, 3, 6, and 8, and overlaps with the binding sites for the substrates - dopamine and amphetamine - as well as for benztropine-like inhibitors. A buried binding pocket was demonstrated by trapping the bound radiolabeled cocaine analogue [3H]CFT in the transporter in two ways. First, through cysteine-mediated cross-linking, and second through an engineered Zn2+ binding site situated above the predicted common binding pocket and positioned corresponding to the newly described non-competitive binding site for tricyclic antidepressants in LeuT6,7. Our data show the molecular basis for the competitive action of cocaine at DAT, which differs from the mode of interaction of antidepressant binding to LeuT. We also demonstrate in DAT a unique binding mode for cocaine and its analogues, which unlike substrates and benztropine-like inhibitors, produce a conformational rearrangement of the binding site that disrupts a stabilizing hydrogen bond between Tyr156 in TM3 and Asp79 in TM1.