title: Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release.
abstract: The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca(2+)-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects.
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Mattias
authors.initials: M
authors.lastname: Rickhag
authors.affiliation: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229.
authors.forename: William A
authors.initials: WA
authors.lastname: Owens
authors.affiliation: Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University Vienna, A-1090 Vienna, Austria.
authors.forename: Marie-Therese
authors.initials: MT
authors.lastname: Winkler
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Kristine Nørgaard
authors.initials: KN
authors.lastname: Strandfelt
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Mette
authors.initials: M
authors.lastname: Rathje
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark; Laboratory of Neuropsychiatry, Faculty of Health and Medical Sciences, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Sørensen
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Andresen
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Madsen
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Trine Nygaard
authors.initials: TN
authors.lastname: Jørgensen
authors.affiliation: Laboratory of Neuropsychiatry, Faculty of Health and Medical Sciences, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Gitta
authors.initials: G
authors.lastname: Wörtwein
authors.affiliation: Laboratory of Neuropsychiatry, Faculty of Health and Medical Sciences, Department of Neuroscience and Pharmacology, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: David P D
authors.initials: DPD
authors.lastname: Woldbye
authors.affiliation: Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University Vienna, A-1090 Vienna, Austria.
authors.forename: Harald
authors.initials: H
authors.lastname: Sitte
authors.affiliation: Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229.
authors.forename: Lynette C
authors.initials: LC
authors.lastname: Daws
authors.affiliation: Molecular Neuropharmacology Laboratory, Lundbeck Foundation Center for Biomembranes in Nanomedicine, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address: gether@sund.ku.dk.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000661:Amphetamine / Q000009:adverse effects / Q000494:pharmacology*; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D057846:Cell-Penetrating Peptides / Q000378:metabolism / Q000494:pharmacology*; D000697:Central Nervous System Stimulants / Q000009:adverse effects / Q000494:pharmacology*; D003342:Corpus Striatum / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D008297:Male; D051379:Mice; D009043:Motor Activity / Q000187:drug effects; D054731:PDZ Domains; D051571:Protein Kinase C-alpha / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D000096926:Benzenesulfonamides
chemical_list: D000661:Amphetamine; D001596:Benzylamines; D057846:Cell-Penetrating Peptides; D000697:Central Nervous System Stimulants; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D051571:Protein Kinase C-alpha; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides; C496630:Prkca protein, mouse
keywords: Addiction; Dopamine Transporter; Neurotransmitter Transport; Protein Kinases; Protein-Protein Interactions; Scaffold Proteins