Pharmacology of Dopamine Release by Amphetamine

描述（由申请人提供）：多巴胺转运体（DAT）是突触中多巴胺（DA）含量和持续时间的基本调节剂，是精神兴奋药物安非他明（AMPH）的主要作用部位。信号转导机制，特别是改变蛋白激酶C （PKC）的信号转导机制，通过直接磷酸化和改变转运，深刻地改变了DAT的活性。短时间暴露于PKC激活剂可引起DAT介导的外排。本提案的总体目标是通过磷酸化检查amph诱导的DA外排通过DAT的调节。假设PKC对DAT的作用可以通过考虑特定PKC同工酶和时间来区分，并且内流和外排可以独立调节。具体目的1：验证PKCbeta，特别是PKCbetaII，促进amph诱导的DA外排的假设。PKC同工酶敲除小鼠和针对特定PKC同工酶的shRNA将区分PKCbetaII、PKCbetaI和PKCalpha对amph刺激的DA外排的影响。将针对PKC同工酶的活性shrna掺入慢病毒并注射到大鼠腹侧被。测量大鼠amph刺激下的DA外排和amph刺激下的运动行为。具体目标2。利用共聚焦显微镜和全内反射荧光显微镜（TIRFM）的生物化学和细胞生物学方法，我们将验证AMPH能快速增加质乳膜上含有dat的囊泡的假设。这一假设将在大鼠原代神经元培养物和内源性含有EGFP-hDAT的小鼠神经元培养物中进行验证。PKCa在AMPH的快速效应中起作用的假设将被验证。具体目标3。DAT中的T62对DA外排很重要，其磷酸化状态改变了内向转运体对DA的亲和力，这一假设将得到验证。我们将研究T62在AMPH作用下被磷酸化的可能性。将使用T62D-DAT和T62A-DAT突变体来研究T62在数据传输中的作用。

DESCRIPTION (provided by applicant): 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.