The dopamine transporter's lipid interactions: understanding transporter function

多巴胺转运蛋白的脂质相互作用：了解转运蛋白的功能

• 批准号: 8808750
• 负责人: AURELIO GALLI
• 金额: $ 33.93万
• 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: 2014-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8808750
• 关键词: Action Potentials; Adopted; Affect; Amphetamine Abuse; Amphetamines; Animal Model; Behavior; Behavioral; Behavioral Model; Binding Sites; Biology; Cell membrane; Cell physiology; Cocaine; Computer Simulation; Data; Disease; Distal; Dopamine; Drosophila genus; Drosophila melanogaster; Electrostatics; Event; Family; Family member; Goals; Health; Homeostasis; Human; Ion Channel; Ions; Lipids; Locomotion; Membrane; Modification; Molecular; Molecular Conformation; Molecular Target; Movement; N Domain; N-terminal; Nature; Neurons; Neurotransmitters; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phospholipids; Phosphorylation; Plasma; Post-Translational Protein Processing; Property; Proteins; Research; Rewards; Role; Signaling Molecule; System; Testing; Translating; cofactor; dopamine transporter; dopaminergic neuron; extracellular; fly; in vivo; member; models and simulation; neurotransmission; new therapeutic target; novel; psychostimulant; reuptake; symporter; 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 have shown that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux, but does not regulate DA uptake. Furthermore, our preliminary studies suggest that the DAT N-terminus interacts electrostatically with phosphatidylinositol-4,5-bisphosphate PIP2 (a key phospholipid enriched at the inner leaflet of the plasma membrane), and that this interaction impairs DA efflux, but not uptake. Our mechanistic hypothesis is that upon phosphorylation, the DAT N-terminus uncouples from PIP2 to disengage from the membrane. Both of these events are required to elicit the DA efflux produced by AMPH. We propose to test our hypothesis and its implications for the behavioral effects of AMPH in vivo, through the following specific aims: 1) To determine the nature of the interaction between DAT N-terminus and PIP2, and describe it in a structural context provided by computational modeling; 2) To determine the role of N-terminus phosphorylation in regulating how DAT and PIP2 interact, and its effect on DAT function; 3) To determine the role of DAT/PIP2 interactions in AMPH-induced behaviors in Drosophila melanogaster. In this animal model, we have established that locomotion is a DAT-dependent 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 findings from specific aims 1 and 2. This will allow us to understand how hDAT N-terminus phosphorylation and associations with PIP2 determine AMPH-induced behaviors. The long-term goals of this research are to understand how AMPH-induced DA efflux and its associated behaviors are dictated by the interactions of hDAT N-terminus with PIP2, and/or by phosphorylation of the N- terminus. By specifically impairing DA efflux, but not uptake, we will determine the contribution of DA efflux in AMPH behaviors. The intent is to uncover novel targets for the treatment of AMPH abuse. From a broad perspective of transporter biology, we will reveal how a DAT structural domain (the N-terminus) via its interactions with the plasma membrane dictates different aspects of transport function.

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。我们已经表明，DAT N-末端磷酸化在五个最远端丝氨酸是必需的AMPH诱导的DA流出，但不调节DA摄取。此外，我们的初步研究表明，DAT N-末端与磷脂酰肌醇-4，5-二磷酸PIP 2（质膜内叶富集的关键磷脂）静电相互作用，这种相互作用损害DA流出，但不摄取。 我们的机制假设是，磷酸化后，DAT N-末端与PIP 2解偶联，从膜上脱离。这两个事件都是引起AMPH产生的DA外排所必需的。我们拟通过以下具体目标来验证我们的假设及其对AMPH在体内行为效应的影响：1）确定DAT N端与PIP 2之间相互作用的性质，并在计算机模拟提供的结构背景下描述它：2）确定N端磷酸化在调节DAT与PIP 2相互作用中的作用，以及它对DAT功能的影响; 3）研究DAT/PIP 2相互作用在AMPH诱导的果蝇行为中的作用。在这个动物模型中，我们已经建立了运动是一个DAT依赖性的行为，并刺激AMPH。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发现，从具体的目标1和2。这将使我们能够了解hDAT N-末端磷酸化和与PIP 2的关联如何决定AMPH诱导的行为。 本研究的长期目标是了解AMPH诱导的DA外排及其相关行为如何由hDAT N-末端与PIP 2的相互作用和/或由N-末端的磷酸化决定。通过特异性地损害DA外排，而不是摄取，我们将确定DA外排在AMPH行为中的贡献。目的是发现治疗AMPH滥用的新靶点。从转运蛋白生物学的广泛角度来看，我们将揭示DAT结构域（N-末端）如何通过其与质膜的相互作用决定转运功能的不同方面。