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

• 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
• 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

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）通过突触释放的DA的主动再摄取来控制DA稳态和神经传递。DAT是负责苯丙胺（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DAT底物，促进DA转运的逆转，从而导致DA通过DAT流出。这种流出导致细胞外DA水平增加，这是AMPH的精神兴奋特性的重要事件。 DAT的N-末端是AMPH引起DA外排的关键结构域。我们证明，DAT N-末端磷酸化在五个最远端丝氨酸是必需的AMPH诱导DA流出。可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白突触融合蛋白1（Stx 1）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。Stx 1在Ser 14被酪蛋白激酶2（CK 2）1磷酸化，我们假设这一事件是由AMPH促进的，并调节DAT-Stx 1的关联。最后，我们发现磷脂酰肌醇-4，5-二磷酸（PIP 2）（我们显示与Stx 1相互作用的辅因子）直接与DAT N-末端相互作用，在这里我们假设协调Stx 1磷酸化，导致DA流出。我们的机制假设是AMPH诱导的Stx 1磷酸化介导的CK 2导致DAT N-末端磷酸化。这些事件由DAT-PIP 2交互协调，并且需要传输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）定义DAT-Stx 1缔合在基础和AMPH条件下是如何协调的; 2）确定Stx 1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，我们在果蝇中开发了一个行为模型。在这个系统中，我们已经建立了运动是DAT调节的行为，并由AMPH刺激。果蝇DA神经元中的Drosophila DAT（dDAT）的缺失抑制AMPH诱导的运动，这种作用通过这些dDAT缺陷的DA神经元中的人DAT（hDAT）的表达而恢复。使用这种策略，我们将在体内翻译我们的分子观察。我们将阐明CK 2介导的Stx 1磷酸化和Stx 1与DAT N-末端的相互作用如何决定AMPH行为。因此，我们最后的具体目标是：3）确定DAT-Stx 1相互作用在AMPH诱导的行为中的作用。 本研究的长期目标是了解AMPH诱导的DA流出及其相关行为如何由Stx 1磷酸化决定和协调。我们的初步数据支持，我们假设，抑制CK 2功能损害特异性DA流出，但不摄取。因此，我们将学习如何选择性地操纵DAT转运周期的不同方面，并确定DA外排在AMPH行为中的贡献。这将揭示一个新的“可药物化”的目标（CK 2）用于治疗AMPH滥用。