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.
Publication types
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Comparative Study
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Amphetamines / pharmacology*
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Animals
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Animals, Newborn
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Benzylamines / pharmacology
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Biological Transport / drug effects
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Blotting, Western / methods
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinases / pharmacology*
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Cells, Cultured
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Central Nervous System Stimulants / pharmacology*
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Chromatography, High Pressure Liquid / methods
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Dopamine / metabolism*
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Dopamine Plasma Membrane Transport Proteins / chemistry
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Dopamine Plasma Membrane Transport Proteins / genetics
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Dopamine Plasma Membrane Transport Proteins / metabolism*
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Enzyme Inhibitors / pharmacology
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Humans
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Immunohistochemistry / methods
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Immunoprecipitation / methods
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In Vitro Techniques
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Membrane Potentials / drug effects
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Membrane Potentials / physiology
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Mesencephalon / cytology
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Neurons / drug effects*
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Neurons / physiology
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Patch-Clamp Techniques / methods
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Phosphorylation / drug effects
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Protein Binding / drug effects
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Protein Binding / physiology
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Protein Structure, Tertiary / physiology
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Rats
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Sulfonamides / pharmacology
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Transfection / methods
Substances
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Amphetamines
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Benzylamines
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Central Nervous System Stimulants
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Dopamine Plasma Membrane Transport Proteins
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Enzyme Inhibitors
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Sulfonamides
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KN 93
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Calcium-Calmodulin-Dependent Protein Kinases
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Dopamine