Probing mechanisms of amphetamine action at plasma membrane and vesicular transporters in vitro and in vivo

体外和体内苯丙胺对质膜和囊泡转运蛋白作用的探讨机制

• 批准号: 9311046
• 负责人: Jonathan A Javitch
• 金额: $ 53.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9311046
• 关键词: Active Biological Transport; Acute; Affect; Amphetamine Abuse; Amphetamines; Attention deficit hyperactivity disorder; Behavior; Behavioral; Behavioral Model; Biochemistry; Biological Assay; Biological Models; Biophysics; Brain; Brain imaging; Cell membrane; Cells; Charge; Computer Analysis; Computer Simulation; Coupled; Cytoplasm; Data; Distal; Dopamine; Drosophila genus; Electrostatics; Elements; Employee Strikes; Event; Goals; Human; Image Analysis; In Vitro; Lead; Length; Lipids; Mediating; Medical; Membrane; Membrane Lipids; Mental disorders; Methamphetamine; Molecular; Molecular Models; Monitor; Mutation; N-terminal; Neurotransmitters; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Physiological; Physiology; Process; Property; Proteins; Psychiatric therapeutic procedure; Rattus; Regulation; Reporting; Role; Self Administration; Serine; Societies; Structure; Synaptic Vesicles; System; Testing; Time; Transmembrane Domain; Validation; Vesicle; Work; amphetamine use; antiport; base; behavioral study; biochemical tools; biophysical techniques; biophysical tools; computerized tools; design; dopamine transporter; fly; in vivo; innovation; insight; molecular dynamics; molecular modeling; multiphoton imaging; novel; pH gradient; prototype; psychostimulant; serotonin transporter; single molecule; single-molecule FRET; uptake; vesicular monoamine transporter

Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. They are known to cause mobilization of cytoplasmic dopamine (DA) to the cell exterior via DA transporter (DAT)-mediated efflux, yet the mechanisms that mediate these actions remain poorly defined and are a focus of this proposal. Using heterologous expression systems and a Drosophila behavioral model, we have shown that AMPH-induced DA efflux and consequent behaviors, but not DA uptake, are dependent on N-terminal phosphorylation of DAT. Our team has also made critical advances in understanding the molecular mechanisms of substrate uptake by studying the bacterial transporter LeuT as a prototype, using state-of-the-art single-molecule approaches and computational analyses. Although the N-terminal region is essentially absent in LeuT and was truncated in the Drosophila DAT (dDAT) structures, our team has reported a computational model of the N terminus of the human DAT (hDAT) from ab initio structure prediction in combination with extensive atomistic molecular dynamics simulations. The analysis shows the N terminus to be highly dynamic, to contain secondary structure elements, and to interact with lipid membranes through electrostatic interactions. Here we aim to probe these structural elements to gain insight into the physiology of DAT and its regulation by AMPHs, using our team's synergistic behavioral, biochemical, biophysical, and computational tools. In parallel studies we aim to explore the mechanisms that regulate AMPH-induced release of DA from synaptic vesicles into the cytoplasm. Using multiphoton imaging of living Drosophila brain we have shown that at pharmacologically relevant concentrations, AMPHs must be actively transported both by DAT and by the vesicular monoamine transporter VMAT in order to diminish the vesicular pH gradient and redistribute vesicular contents. Still, how these events lead to redistribution of DA to the cytoplasm remains unknown. Recent data suggest that VMAT N-terminal phosphorylation is essential for AMPH-induced DA efflux from vesicles, and we propose to explore this hypothesis mechanistically and test it in vivo. Our established multi-scale approach integrates biochemistry and biophysics of purified proteins, single-molecule FRET and computational analysis, with cell-based assays, Drosophila brain imaging, analysis of in vivo phosphorylation, and behavioral studies in living flies to probe the role of DAT and VMAT in the actions of AMPHs in the appropriate physiological and structural contexts, in the following SPECIFIC AIMs: AIM 1. To elucidate the role of membrane interactions in modulating phosphorylation of the N terminus of DAT and its ability to mediate AMPH-induced DA efflux and behaviors. AIM 2. To determine how N-terminal phosphorylation alters DAT function and dynamics. AIM 3. To determine the role of VMAT and its putative N-terminal phosphorylation in AMPH-induced DA efflux from synaptic vesicles in vivo and in vitro. This work will provide a clear validation of novel targets for medications that block AMPH action through mechanisms that do not alter DA uptake.

苯丙胺（AMPHS）是广泛使用和滥用的有效的心理刺激物，并具有深刻的医学 和社会影响。已知它们会导致细胞质多巴胺（DA）动员到细胞外部 通过DA转运蛋白（DAT）介导的外排，但介导这些作用的机制仍然很差 定义，是该提议的重点。使用异源表达系统和果蝇行为 模型，我们已经表明，Amph诱导的DA外排和随之而来的行为，但不吸收DA是 取决于DAT的N末端磷酸化。我们的团队也在理解方面取得了重要的进步 通过研究细菌转运蛋白Leut作为原型的底物摄取的分子机制，使用 最先进的单分子方法和计算分析。尽管N末端区域是 本质上是在Leut中没有的，在果蝇DAT（DDAT）结构中被截断，我们的团队报告了 从头算结构预测中人类DAT（HDAT）N末端的计算模型 结合广泛的原子分子动力学模拟。分析表明N末端到 高度动态，包含二级结构元素，并与脂质膜相互作用 静电相互作用。在这里，我们旨在探究这些结构元素，以深入了解 DAT及其对AMPHS的调节，使用我们团队的协同行为，生化，生物物理和 计算工具。在平行研究中，我们旨在探索调节Amph诱导释放的机制 从突触囊泡进入细胞质的DA。使用活果蝇大脑的多光子成像我们有 表明，在药理学相关的浓度下，AMPH必须由DAT主动运输 并通过囊泡单胺转运蛋白VMAT，以减少囊泡pH梯度和 再分配囊泡含量。尽管如此，这些事件如何导致DA重新分布到细胞质 未知。最近的数据表明，VMAT N末端磷酸化对于AMPH诱导的DA外排是必不可少的 从囊泡中，我们提议从机械上探索这一假设并在体内对其进行测试。我们建立的 多尺度方法整合了纯化蛋白质，单分子fret和的生物化学和生物物理学 计算分析，基于细胞的测定，果蝇脑成像，体内磷酸化的分析， 和生命中的行为研究以探究DAT和VMAT在AMPHS在AMPH中的作用的作用 适当的生理和结构背景，以下特定目的：目的1。阐明 膜相互作用在调节D DAT的N末端的磷酸化及其介导能力的能力中 Amph引起的DA外排和行为。目标2。确定N末端磷酸化如何改变DAT 功能和动力学。目标3。确定VMAT及其推定的N末端磷酸化的作用 Amph诱导的体内和体外突触囊泡的DA外排。这项工作将为明确的验证 通过不会改变DA摄取的机制阻止AMPH作用的药物的新靶标。