title: Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.
abstract: The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.
authors.affiliation: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, NY 10032, USA; Center for Molecular Biology and Neuroscience, Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, PO Box 1105 Blindern, N 0317 Oslo, Norway. Electronic address: yvette.dehnes@h-lab.no.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Dehnes
authors.affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA. Electronic address: jufang.shan@gmail.com.
authors.forename: Jufang
authors.initials: J
authors.lastname: Shan
authors.affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA; Schrodinger, Inc., 120 West 45th Street, New York, NY 10036, USA. Electronic address: thijs.beuming@schrodinger.com.
authors.forename: Thijs
authors.initials: T
authors.lastname: Beuming
authors.affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA; HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, 1305 York Avenue, New York, NY 10021, USA. Electronic address: les2007@med.cornell.edu.
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.affiliation: Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA; HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Medical College of Cornell University, 1305 York Avenue, New York, NY 10021, USA. Electronic address: haw2002@physbio-tech.net.
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.affiliation: Center for Molecular Recognition, Columbia University College of Physicians and Surgeons, 630 W. 168th, New York, NY 10032, USA; Department of Psychiatry & Pharmacology, Columbia University College of Physicians and Surgeons, 1051 Riverside Drive, New York, NY 10032, USA; Division of Molecular Therapeutics, New York State Psychiatric Institute, 1051 Riverside Drive, New York, NY 10032, USA. Electronic address: jaj2@columbia.edu.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
mesh_terms: D003001:Cloning, Molecular; D003042:Cocaine / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D011487:Protein Conformation; D021381:Protein Transport; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014326:Tropanes / Q000378:metabolism / Q000494:pharmacology; D014439:Tyramine / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
chemical_list: C434646:2-carbomethoxy-3-(3',4'-dichlorophenyl)tropane; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D013439:Sulfhydryl Reagents; D014326:Tropanes; D003042:Cocaine; D015032:Zinc; D003545:Cysteine; D004298:Dopamine; D014439:Tyramine
keywords: Cocaine binding; Cysteine accessibility; Dopamine transport; Dopamine transporter (DAT); Molecular dynamics simulations