The Role of the Dopamine Transporter in Psychostimulant Abuse

描述(申请人提供)：多巴胺(DA)转运体(DAT)通过主动重新摄取突触释放的DA来控制DA的动态平衡和神经传递。DAT是苯丙胺(Amph)和可卡因的有益特性和滥用潜力的主要分子靶标。AMPH作为DAT底物，促进DA转运的逆转，从而导致DA通过DAT外流。这种外流导致细胞外DA水平的增加，这对AMPHs的精神运动刺激性是一个重要的事件。DAT的N-末端是一个结构域，对Amph导致DA外流至关重要。我们证明了AMPH诱导的DA外流需要5个最远端Ser上的DAT N-末端的磷酸化。可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)蛋白Synaxin1(STX1)与DAT的N末端相互作用，这种相互作用支持Amph导致DA外流的能力。STX1在Ser14位被酪蛋白激酶2(CK2)1磷酸化，我们推测这一事件是由AMPH促进的，并调节DAT-STX1结合。最后，我们发现磷脂酰肌醇-4，5-二磷酸(PIP2)(我们发现的一种与STX1相互作用的辅助因子)直接与DAT的N末端相互作用，在这里我们假设STX1是磷酸化的，导致DA外流。我们的机制假设是Amph诱导的由CK2介导的STX1磷酸化导致DAT N末端的磷酸化。这些事件由DAT-PIP2相互作用协调，并需要传递AMPH的动作。我们建议通过以下具体目标来验证我们的假设：1)确定DAT-STX1在基础条件和AMPH条件下如何协调；2)确定STX1磷酸化在AMPH诱导的DA外流中的作用。为了测试我们在体内的分子发现，我们开发了一个在黑腹果蝇身上的行为模型。在这个系统中，我们已经确定了运动是一种DAT调节的行为，并受到AMPH的刺激。果蝇DA神经元中果蝇DAT(DDAT)的缺失可抑制AMPH诱导的运动，这种作用可通过在这些缺失dDAT的DA神经元中表达人DAT(HDAT)而恢复。使用这一策略，我们将在体内转换我们的分子观察。我们将阐明CK2介导的STX1磷酸化和STX1与DAT N末端的相互作用如何决定AMPH行为。因此，我们的最后一个具体目标是：3)确定DAT-STX1相互作用在AMPH诱导的行为中的作用。这项研究的长期目标是了解Amph诱导的DA外流及其相关行为是如何通过STX1磷酸化来决定和协调的。在我们的初步数据支持下，我们假设抑制CK2功能会特异性地损害DA的外流，但不会损害摄取。因此，我们将学习如何有选择地操纵DAT运输周期的不同方面，并确定DA外流在AMPH行为中的贡献。这将发现一个新的治疗AMPH滥用的“可用药”靶点(CK2)。

DESCRIPTION (provided by applicant): The dopamine (DA) transporter (DAT) controls DA homeostasis and neurotransmission by the active reuptake of synaptically released DA. The DAT is the major molecular target responsible for the rewarding properties and the abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, promoting the reversal of DA transport, thereby resulting in DA efflux via DAT. This efflux leads to increased extracellular DA levels, an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain that is critical for AMPH to cause DA efflux. We demonstrated that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin1 (Stx1) interacts with the DAT N-terminus and this interaction supports the ability of AMPH to cause DA efflux. Stx1 is phosphorylated at Ser14 by casein kinase 2 (CK2)1, an event we hypothesize is promoted by AMPH and regulates DAT-Stx1 association. Finally, we discovered that phosphatidylinositol-4,5-bisphosphate (PIP2) (a cofactor we show interacts with Stx1) directly interacts with the DAT N-terminus, and here we hypothesize coordinates Stx1 phosphorylation, leading to DA efflux. Our mechanistic hypothesis is that AMPH-induced Stx1 phosphorylation mediated by CK2 leads to DAT N-terminus phosphorylation. These events are coordinated by DAT-PIP2 interaction and are required to transmit the actions of AMPH. We propose to test our hypothesis through the following specific aims: 1) To define how DAT-Stx1 association is coordinated under basal and AMPH conditions; and 2) To determine the role of Stx1 phosphorylation in AMPH-induced DA efflux. To test our molecular discoveries in vivo, we have developed a behavioral model in Drosophila melanogaster. In this system, we have established that locomotion is a DAT-regulated behavior, and is stimulated by AMPH. Deletion of Drosophila DAT (dDAT) in DA neurons of flies inhibits AMPH-induced locomotion, an effect that is restored by the expression of the human DAT (hDAT) in these dDAT-deficient DA neurons. Using this strategy, we will translate in vivo our molecular observations. We will elucidate how CK2-mediated Stx1 phosphorylation and Stx1 interaction with the DAT N-terminus determine AMPH behaviors. Thus, our last specific aim is: 3) To determine the role of DAT-Stx1 interactions in AMPH-induced behaviors. The long-term goal of this research is to understand how AMPH-induced DA efflux and its associated behaviors are dictated and coordinated by Stx1 phosphorylation. Supported by our preliminary data, we hypothesize that inhibiting CK2 function impairs specifically DA efflux, but not uptake. Thus, we will learn how to selectively manipulate different aspects of the DAT transport cycle and determine the contribution of DA efflux in AMPH behaviors. This will uncover a new "druggable" target (CK2) for the treatment of AMPH abuse.