The Role of the Dopamine Transporter in Psychostimulant Abuse

多巴胺转运蛋白在精神兴奋剂滥用中的作用

基本信息

批准号：
9330133
负责人：
AURELIO GALLI
金额：
$ 11.44万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9330133
关键词：
Amphetamine Abuse Amphetamines Animal Model Antidepressive Agents Behavior Behavioral Behavioral Model Carrier Proteins Cell membrane Cell physiology Cocaine Collaborations Data Disease Distal Dopamine Drosophila genus Drosophila melanogaster Event Family member Functional disorder Goals Health Homeostasis Human Impairment Learning Lipids Locomotion Mediating Membrane Membrane Microdomains Membrane Transport Proteins Molecular Molecular Target Movement N-terminal Nature Neurons Organism Pharmaceutical Preparations Phosphatidylinositol 4,5-Diphosphate Phospholipids Phosphorylation Physiological Plasma Property Proteins Recruitment Activity Research Research Personnel Rewards Role SNAP receptor Signaling Molecule System Testing Time Translating behavior observation calmodulin-dependent protein kinase II casein kinase II cofactor dopamine transporter dopaminergic neuron effective therapy experimental study extracellular fly in vivo knockout gene neuronal circuitry neurotransmission new therapeutic target noradrenaline transporter psychostimulant public health relevance receptor reuptake serotonin transporter social stimulant abuse uptake

项目摘要

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.

描述（由申请人提供）：多巴胺（DA）转运蛋白（DAT）通过主动重摄取突触释放的 DA 来控制 DA 稳态和神经传递。 DAT 是导致安非他明 (AMPH) 和可卡因的有益特性和滥用潜力的主要分子靶点。 AMPH 作为 DAT 底物，促进 DA 转运的逆转，从而导致 DA 通过 DAT 流出。这种外流导致细胞外 DA 水平增加，这对于 AMPH 的精神运动兴奋特性非常重要。 DAT 的 N 末端是一个对于 AMPH 引起 DA 流出至关重要的结构域。我们证明了 AMPH 诱导的 DA 流出需要 DAT N 末端 5 个最远端 Ser 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 Syntaxin1 (Stx1) 与 DAT N 末端相互作用，这种相互作用支持 AMPH 引起 DA 流出的能力。 Stx1 在 Ser14 位点被酪蛋白激酶 2 (CK2)1 磷酸化，我们假设这一事件由 AMPH 促进并调节 DAT-Stx1 关联。最后，我们发现磷脂酰肌醇-4,5-二磷酸 (PIP2)（我们证明与 Stx1 相互作用的辅助因子）直接与 DAT N 末端相互作用，在这里我们假设协调 Stx1 磷酸化，导致 DA 外流。我们的机制假设是 CK2 介导的 AMPH 诱导的 Stx1 磷酸化导致 DAT N 末端磷酸化。这些事件通过 DAT-PIP2 相互作用进行协调，并且需要传输 AMPH 的操作。我们建议通过以下具体目标来检验我们的假设：1）定义DAT-Stx1关联在基础和AMPH条件下如何协调； 2) 确定 Stx1 磷酸化在 AMPH 诱导的 DA 流出中的作用。为了在体内测试我们的分子发现，我们开发了果蝇的行为模型。在这个系统中，我们已经确定运动是 DAT 调节的行为，并受到 AMPH 的刺激。果蝇 DA 神经元中果蝇 DAT (dDAT) 的缺失会抑制 AMPH 诱导的运动，这种效应可以通过这些 dDAT 缺陷的 DA 神经元中人类 DAT (hDAT) 的表达来恢复。使用这种策略，我们将在体内转化我们的分子观察结果。我们将阐明 CK2 介导的 Stx1 磷酸化以及 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 诱导行为中的作用。这项研究的长期目标是了解 STx1 磷酸化如何决定和协调 AMPH 诱导的 DA 外流及其相关行为。根据我们的初步数据，我们假设抑制 CK2 功能会具体损害 DA 流出，但不会损害摄取。因此，我们将学习如何选择性地操纵 DAT 转运循环的不同方面，并确定 DA 外流在 AMPH 行为中的贡献。这将揭示治疗 AMPH 滥用的新“可成药”靶点 (CK2)。