The dopamine transporter (DAT) is the fundamental regulator of dopamine (DA) content and duration in the synapse, and is a predominant site of action of the psychostimulant drug, amphetamine (AMPH). Signal transduction mechanisms, especially ones altering protein kinase C (PKC), profoundly alter DAT activity through direct phosphorylation and by altering trafficking. Short exposures to PKC activators elicit DAT- mediated efflux. The overall goal of this proposal is to examine the regulation of AMPH-induced DA efflux through DAT by phosphorylation. The hypothesis will be tested that actions of PKC on DAT can be distinguished by a consideration of specific PKC isozymes and by time and that influx and efflux can be regulated independently.
Specific Aim 1 : Test the hypothesis that PKCbeta, particularly PKCbetaII, promotes AMPH-induced DA efflux. PKC isozyme knockout mice and shRNA against specific PKC isozymes will differentiate the effects of PKCbetaII, PKCbetaI and PKCalpha on AMPH-stimulated DA efflux. Active shRNAs against the PKC isozymes will be incorporated into a lentivirus and injected into rat ventral tegmentum. AMPH-stimulated DA efflux and AMPH-stimulated locomotor behavior will be measured in the rats.
Specific Aim 2. Using biochemical and cell biological approaches with confocal microscopy and total internal reflectance fluorescent microscopy (TIRFM), we will test the hypothesis that AMPH has a rapid effect to increase the DAT-containing vesicles at the plasmalemmal membrane. This hypothesis will be tested in rat primary neuronal cultures and neuronal cultures from mice endogenously containing EGFP-hDAT. The hypothesize that PKCa plays a role in the rapid effects of AMPH will be tested.
Specific Aim 3. The hypothesis that T62 in DAT is important for DA efflux and that its phosphorylation status alters the affinity for DA at the inward-facing transporter will be tested. The possibility that T62 is phosphorylated in response to AMPH will be examined. The role of T62 in DAT trafficking will be investigated using the T62D-DAT and T62A-DAT mutants. The initial action of AMPH at the dopamine transporter initiates a series of events that can lead to drug addiction. Knowledge of the mechanism of action and regulation of these transporters are integral to understanding the regulation of synaptic dopamine and controlling the action of AMPH. In addition, acute AMPH serves as a useful model for mania so knowledge of AMPH's mechanism of action could aid in understanding of that disease.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project (R01)
Project #
Application #
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Lin, Geraline
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
Zip Code
Zestos, Alexander G; Carpenter, Colleen; Kim, Youngsoo et al. (2018) Ruboxistaurin Reduces Cocaine-Stimulated Increases in Extracellular Dopamine by Modifying Dopamine-Autoreceptor Activity. ACS Chem Neurosci :
Mikelman, Sarah R; Guptaroy, Bipasha; Gnegy, Margaret E (2017) Tamoxifen and its active metabolites inhibit dopamine transporter function independently of the estrogen receptors. J Neurochem 141:31-36
Carpenter, Colleen; Zestos, Alexander G; Altshuler, Rachel et al. (2017) Direct and Systemic Administration of a CNS-Permeant Tamoxifen Analog Reduces Amphetamine-Induced Dopamine Release and Reinforcing Effects. Neuropsychopharmacology 42:1940-1949
Mikelman, Sarah; Mardirossian, Natalie; Gnegy, Margaret E (2017) Tamoxifen and amphetamine abuse: Are there therapeutic possibilities? J Chem Neuroanat 83-84:50-58
Carpenter, Colleen; Sorenson, Roderick J; Jin, Yafei et al. (2016) Design and synthesis of triarylacrylonitrile analogues of tamoxifen with improved binding selectivity to protein kinase C. Bioorg Med Chem 24:5495-5504
Singer, Bryan F; Guptaroy, Bipasha; Austin, Curtis J et al. (2016) Individual variation in incentive salience attribution and accumbens dopamine transporter expression and function. Eur J Neurosci 43:662-70
Zestos, Alexander G; Mikelman, Sarah R; Kennedy, Robert T et al. (2016) PKC? Inhibitors Attenuate Amphetamine-Stimulated Dopamine Efflux. ACS Chem Neurosci 7:757-66
Luderman, Kathryn D; Chen, Rong; Ferris, Mark J et al. (2015) Protein kinase C beta regulates the D?-like dopamine autoreceptor. Neuropharmacology 89:335-41
Fraser, Rheaclare; Chen, Yongyue; Guptaroy, Bipasha et al. (2014) An N-terminal threonine mutation produces an efflux-favorable, sodium-primed conformation of the human dopamine transporter. Mol Pharmacol 86:76-85
Bosse, Kelly E; Jutkiewicz, Emily M; Schultz-Kuszak, Kristin N et al. (2014) Synergistic activity between the delta-opioid agonist SNC80 and amphetamine occurs via a glutamatergic NMDA-receptor dependent mechanism. Neuropharmacology 77:19-27

Showing the most recent 10 out of 29 publications