The Role of the Dopamine Transporter in Psychostimulant Abuse

描述（由申请人提供）：多巴胺（DA）转运体（DAT）通过主动再摄取突触释放的DA来控制DA稳态和神经传递。DAT是安非他明（AMPH）和可卡因的奖励特性和滥用潜力的主要分子靶标。AMPH作为数据底物，促进数据转运的逆转，从而导致数据通过数据外排。这种外排导致细胞外DA水平增加，这对于amph的精神运动兴奋特性是一个重要的事件。DAT的n端是一个结构域，对AMPH引起DA外排至关重要。我们证明了在amph诱导的DA外排中，DAT n端在最末端的5个丝氨酸磷酸化是必需的。可溶性n -乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白syntaxin1 （Stx1）与DAT n端相互作用，这种相互作用支持AMPH引起DA外排的能力。Stx1在Ser14位点被酪蛋白激酶2 (CK2)1磷酸化，我们假设这一事件是由AMPH促进的，并调节DAT-Stx1的关联。最后，我们发现磷脂酰肌醇-4,5-二磷酸（PIP2）（我们发现与Stx1相互作用的辅助因子）直接与DAT n端相互作用，这里我们假设坐标Stx1磷酸化，导致DA外排。我们的机制假设是amph诱导的CK2介导的Stx1磷酸化导致DAT n端磷酸化。这些事件由DAT-PIP2相互作用协调，是传递AMPH动作所必需的。我们提出通过以下具体目标来验证我们的假设：1)定义DAT-Stx1关联在基础和AMPH条件下是如何协调的；2)确定Stx1磷酸化在amph诱导的DA外排中的作用。为了在体内测试我们的分子发现，我们在黑腹果蝇身上建立了一个行为模型。在这个系统中，我们已经确定运动是一种由dat调节的行为，并受到AMPH的刺激。果蝇DA神经元中Drosophila DAT （dDAT）的缺失可以抑制amph诱导的运动，而在这些缺乏dDAT的DA神经元中表达人类DAT （hDAT）可以恢复这种作用。使用这种策略，我们将在体内翻译我们的分子观察结果。我们将阐明ck2介导的Stx1磷酸化和Stx1与DAT n端相互作用如何决定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诱导行为中的作用。本研究的长期目标是了解amph诱导的DA外排及其相关行为是如何由Stx1磷酸化决定和协调的。根据我们的初步数据，我们假设抑制CK2功能特异性地损害DA外排，但不损害摄取。因此，我们将学习如何有选择地操纵数据传输周期的不同方面，并确定数据外排在AMPH行为中的贡献。这将揭示治疗AMPH滥用的一个新的“可药物”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.