Pharmacology of Dopamine Release by Amphetamine

描述(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器，也是精神刺激药物苯丙胺(AMPH)的主要作用部位。信号转导机制，尤其是那些改变蛋白激酶C(PKC)的机制，通过直接磷酸化和改变运输，深刻地改变DAT的活性。短时间暴露于PKC激活剂可诱导DAT介导的外流。这项建议的总体目标是研究AMPH通过磷酸化DAT对DA外流的调节。这一假设将得到检验，即PKC对DAT的作用可以通过考虑特定的PKC同工酶和时间来区分，并且流入和流出可以独立调节。具体目的1：验证PKCbeta，尤其是PKCbetaII促进Amph诱导的DA外流的假设。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在Amph刺激的DA外流中的作用。针对PKC同工酶的活性shRNAs将被整合到慢病毒中并注射到大鼠腹侧被盖中。将测量AMPH刺激的DA外流和AMPH刺激的运动行为。具体目的2.利用生化和细胞生物学方法，结合共聚焦显微镜和全内反射荧光显微镜(TIRFM)，我们将验证Amph具有快速增加质膜上含DAT的囊泡的假设。这一假设将在大鼠原代神经元培养和内源性含有EGFP-HDAT的小鼠神经元培养中进行验证。PKCA在Amph的快速效应中发挥作用的假设将得到检验。具体目的3.假设DAT中的T62对DA外流很重要，并且它的磷酸化状态改变了内向转运体与DA的亲和力，这一假设将得到验证。将检查T62被磷酸化以响应AMPH的可能性。将使用T62D-DAT和T62A-DAT突变体来研究T62在DAT运输中的作用。 Amph在多巴胺转运体的最初作用启动了一系列可能导致药物成瘾的事件。了解这些转运蛋白的作用机制和调控对于理解突触多巴胺的调节和控制AMPH的作用是不可或缺的。此外，急性AMPH是治疗躁狂症的有用模型，因此了解AMPH的作用机制有助于了解这种疾病。

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.