title: Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport.
abstract: Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the distal C terminus of DAT and colocalized with DAT in dopaminergic neurons. CaMKIIalpha stimulated dopamine efflux via DAT in response to amphetamine in heterologous cells and in dopaminergic neurons. CaMKIIalpha phosphorylated serines in the distal N terminus of DAT in vitro, and mutation of these serines eliminated the stimulatory effects of CaMKIIalpha. A mutation of the DAT C terminus impairing CaMKIIalpha binding also impaired amphetamine-induced dopamine efflux. An in vivo role for CaMKII was supported by chronoamperometry measurements showing reduced amphetamine-induced dopamine efflux in response to the CaMKII inhibitor KN93. Our data suggest that CaMKIIalpha binding to the DAT C terminus facilitates phosphorylation of the DAT N terminus and mediates amphetamine-induced dopamine efflux.
authors.affiliation: Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
authors.forename: Jacob U
authors.initials: JU
authors.lastname: Fog
authors.forename: Habibeh
authors.initials: H
authors.lastname: Khoshbouei
authors.forename: Marion
authors.initials: M
authors.lastname: Holy
authors.forename: William A
authors.initials: WA
authors.lastname: Owens
authors.forename: Christian Bjerggaard
authors.initials: CB
authors.lastname: Vaegter
authors.forename: Namita
authors.initials: N
authors.lastname: Sen
authors.forename: Yelyzaveta
authors.initials: Y
authors.lastname: Nikandrova
authors.forename: Erica
authors.initials: E
authors.lastname: Bowton
authors.forename: Douglas G
authors.initials: DG
authors.lastname: McMahon
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Colbran
authors.forename: Lynette C
authors.initials: LC
authors.lastname: Daws
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
authors.forename: Ulrik
authors.initials: U
authors.lastname: Gether
mesh_terms: D000662:Amphetamines / Q000494:pharmacology*; D000818:Animals; D000831:Animals, Newborn; D001596:Benzylamines / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western / Q000379:methods; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000494:pharmacology*; D002478:Cells, Cultured; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008636:Mesencephalon / Q000166:cytology; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D000096926:Benzenesulfonamides
chemical_list: D000662:Amphetamines; D001596:Benzylamines; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000697:Central Nervous System Stimulants; D004298:Dopamine; D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D013449:Sulfonamides; C072105:KN 93; D000096926:Benzenesulfonamides