Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport.
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.
Fog
Jacob U
JU
Molecular Neuropharmacology Group and Center for Pharmacogenomics, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark.
Khoshbouei
Habibeh
H
Holy
Marion
M
Owens
William A
WA
Vaegter
Christian Bjerggaard
CB
Sen
Namita
N
Nikandrova
Yelyzaveta
Y
Bowton
Erica
E
McMahon
Douglas G
DG
Colbran
Roger J
RJ
Daws
Lynette C
LC
Sitte
Harald H
HH
Javitch
Jonathan A
JA
Galli
Aurelio
A
Gether
Ulrik
U
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
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