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）和可卡因的奖励性质和滥用潜力的主要分子靶标。 AMPH充当DAT底物，促进了DA传输的逆转，从而导致DA FEFFLUX通过DAT。这种外排导致细胞外DA水平的增加，这是对AMPHS的精神运动刺激特性的重要性。 DAT的N末端是一个结构域，对于AMPH引起DA外排至关重要。我们已经表明，AMPH诱导的DA外排需要五个最远端Ser的DAT N末端磷酸化，但不调节DA的吸收。此外，我们的初步研究表明，DAT N-末端与磷脂酰肌醇-4,5-双磷酸pip2（一种富含质膜内部传叶的关键磷脂）与磷脂酰肌醇-4,5-双磷酸pip2相互作用，并且这种相互作用会损害DA FEFFLUX，但不会摄入。 我们的机械假设是，在磷酸化后，从PIP2到脱离膜的DAT N-末端是从膜上脱离的。这两个事件都需要引起AMPH产生的DA外排。我们建议通过以下特定目的来检验我们的假设及其对Amph在体内行为影响的影响：1）确定DAT N-Terminus和Pip2之间相互作用的性质，并在计算建模提供的结构环境中进行描述； 2）确定N末端磷酸化在调节DAT和PIP2相互作用及其对DAT功能的影响中的作用； 3）确定dat/pip2相互作用在果蝇果蝇中的AMPH诱导行为中的作用。在这个动物模型中，我们确定运动是一种依赖于DAT的行为，并被AMPH刺激。 DA神经元中果蝇DAT（DDAT）的缺失抑制了Amph诱导的运动，这种效果通过这些DDAT缺乏的DA神经元中人类DAT（HDAT）的表达恢复。使用这种策略，我们将在体内转化我们的分子发现从特定目的1和2转换。这将使我们能够了解HDAT N末端磷酸化以及与PIP2的关联如何确定AMPH诱导的行为。 这项研究的长期目标是了解Amph诱导的DA外排如何取决于HDAT N端与PIP2的相互作用和/或N-末端的磷酸化。通过特别损害DA外排，但不会吸收，我们将确定DA外排在AMPH行为中的贡献。目的是发现治疗Amph滥用的新颖目标。从转运蛋白生物学的广义角度来看，我们将通过其与质膜的相互作用来揭示数据结构域（N端）如何决定运输功能的不同方面。