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.