Understanding the Role of Dopamine Efflux in Amphetamine-induced Behaviors

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作为一种DAT底物，通过一种尚未完全了解的机制，促进DA外排（逆转DAT对DA的运输）进入细胞外空间。细胞外DA水平的增加对于amph的精神运动刺激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外排的能力至关重要。我们已经证明可溶性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。此外，已知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，并促进DAT-Stx1关联，从而支持DA外排。我们的分子假设是AMPH诱导Stx1磷酸化，导致Stx1与DAT n端相关联，从而触发DAT介导的DA外排。我们提出通过以下具体目的来验证我们的分子假设：1)确定CK2和Stx1磷酸化在AMPH诱导的DA外排中的作用。接下来，我们将在体内测试我们的分子发现。我们已经开发了黑腹果蝇作为模型来测试AMPH的行为影响。在这个系统中，我们已经确定了运动是一种由数据调节的行为，并受到AMPH的刺激。果蝇DA神经元中DAT （dDAT）的缺失会抑制AMPH诱导果蝇运动的能力。amph诱导的运动可通过在ddat缺失的DA神经元中表达人DAT （hDAT）来恢复。有了这个策略，我们将把我们的分子观察转化为体内模型，使我们能够测试行为的有效性。我们的体内假设是AMPH诱导的行为（例如运动）依赖于ck2介导的Stx1磷酸化。因此，我们的第二个具体目标是：2)确定CK2和Stx1磷酸化在amph诱导行为中的作用。本研究的长期目标是了解如何有选择地操纵数据传输周期的不同方面，以损害数据外排和AMPH行为。根据我们的初步数据，我们假设抑制CK2功能会特异性地损害dat介导的DA外排，但不会损害dat介导的DA摄取。这项拟议的研究将发现一个新的“可药物”靶点（CK2）治疗AMPH滥用。

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 abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, and, through a mechanism not fully understood, promotes the efflux of DA (reversal of DAT's transport of DA) into the extracellular space. This increase of extracellular DA levels is an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain critical to AMPH's ability to cause DA efflux. We have shown that 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. Additionally, Stx1 is known to be phosphorylated at Ser14 by casein kinase 2 (CK2)1, an event we hypothesize is promoted by AMPH and promotes DAT-Stx1 association supporting, therefore, DA efflux. Our molecular hypothesis is that AMPH induces Stx1 phosphorylation, leading to the association of Stx1 with the DAT N-terminus and thereby triggering DAT-mediated DA efflux. We propose to test our molecular hypothesis through the following specific aim: 1) To determine the roles of CK2 and Stx1 phosphorylation in AMPH- induced DA efflux. Next, we will test our molecular discoveries in vivo. We have developed Drosophila melanogaster as a model to test the behavioral effects of AMPH. 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 inhibits AMPH's ability to induce locomotion in flies. AMPH-induced locomotion is restored by the expression of the human DAT (hDAT) in dDAT-deficient DA neurons. With this strategy, we will translate our molecular observations to an in vivo model, allowing us to test behavioral validity. Our in vivo hypothesis is that AMPH- induced behaviors (e.g., locomotion) are dependent on CK2-mediated Stx1 phosphorylation. Thus, our second specific aim is: 2) To determine the role of CK2 and Stx1 phosphorylation in AMPH-induced behaviors. The long term goal of this research is to learn how to selectively manipulate different aspects of the DAT transport cycle to impair DA efflux and AMPH behaviors. Supported by our preliminary data, we hypothesize that inhibiting CK2 function will specifically impair DAT-mediated DA efflux, but not DAT-mediated DA uptake. This proposed research will uncover a new "druggable" target (CK2) for the treatment of AMPH abuse.