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: 9601228
• 负责人: AURELIO GALLI
• 金额: $ 18.79万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-12-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9601228
• 关键词: dopamine; transporter; lipid; interactions; understanding

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(申请人提供)：多巴胺(DA)转运体(DAT)通过主动重新摄取突触释放的DA来控制DA的动态平衡和神经传递。DAT是苯丙胺(Amph)和可卡因的有益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外流。这种外流导致细胞外DA水平的增加，这对AMPHs的精神运动刺激性是一个重要的事件。DAT的N-末端是一个结构域，对Amph导致DA外流至关重要。我们已经证明，AMPH诱导的DA外流需要5个最远端Ser的DAT N末端的磷酸化，但不调节DA的摄取。此外，我们的初步研究表明，DAT的N端与磷脂酰肌醇-4，5-二磷酸PIP2(一种富含在质膜内叶的关键磷脂)以静电方式相互作用，这种相互作用损害了DA的外流，但不影响摄取。我们的机制假设是，在磷酸化后，DAT的N末端与PIP2解偶联，从膜上脱离。这两个事件都是引发AMPH产生的DA外流所必需的。我们建议通过以下具体目标在体内验证我们的假设及其对amph行为效应的影响：1)确定DAT N末端与PIP2之间相互作用的性质，并在计算模型提供的结构背景下对其进行描述；2)确定N末端磷酸化在调节DAT和PIP2相互作用中的作用及其对DAT功能的影响；3)确定DAT/PIP2相互作用在amph诱导的黑腹果蝇行为中的作用。在这个动物模型中，我们已经建立了运动是一种DAT依赖的行为，并被AMPH刺激。果蝇DA神经元中果蝇DAT(DDAT)的缺失可抑制AMPH诱导的运动，这种作用可通过在这些缺失dDAT的DA神经元中表达人DAT(HDAT)而恢复。使用这一策略，我们将在体内将我们的分子发现从特定的目标1和2转化。这将使我们能够理解HDAT N末端磷酸化及其与PIP2的关联如何决定AMPH诱导的行为。这项研究的长期目标是了解Amph诱导的DA外流及其相关行为是如何通过HDAT N端与PIP2的相互作用和/或N端的磷酸化来决定的。通过专门损害DA外流，但不是摄取，我们将确定DA外流在AMH行为中的贡献。其目的是发现治疗安非他明滥用的新目标。从转运蛋白生物学的广泛角度，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来调控转运蛋白功能的不同方面。