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

苯丙胺类药物是一种广泛使用和滥用的强效精神兴奋剂， 和社会影响。已知它们可引起细胞质多巴胺（DA）向细胞外移动 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 这是本书的重点，也是本书的重点。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流出和随之而来的行为，但不是DA摄取， 依赖于DAT的N-末端磷酸化。我们的团队在理解 以细菌转运蛋白LeuT为原型，利用 最先进的单分子方法和计算分析。虽然N-末端区域是 我们的研究小组报告说，LeuT中基本上不存在，果蝇DAT（dDAT）结构中被截短， 从从头算结构预测的人DAT（hDAT）的N末端的计算模型， 结合广泛的原子分子动力学模拟。分析显示N末端与 是高度动态的，含有二级结构元件，并通过与脂质膜相互作用， 静电相互作用在这里，我们的目标是探索这些结构要素，以深入了解的生理学， DAT和它的调节AMPH，使用我们的团队的协同行为，生物化学，生物物理， 计算工具。在平行研究中，我们的目标是探索调节AMPH诱导释放的机制， 从突触囊泡进入细胞质。利用活体果蝇大脑的多光子成像， 表明，在相关的浓度，AMPH必须积极运输的DAT 和囊泡单胺转运蛋白VMAT，以减少囊泡pH梯度， 重新分配囊泡内容物。尽管如此，这些事件如何导致DA重新分布到细胞质仍然存在 未知最近的数据表明，VMAT N-末端磷酸化是必不可少的AMPH诱导DA流出 从囊泡，我们建议探讨这一假设的机制和测试它在体内。我们既定 多尺度方法整合了纯化蛋白质的生物化学和生物物理学，单分子FRET和 计算分析，基于细胞的分析，果蝇脑成像，体内磷酸化分析， 和行为研究，以探讨DAT和VMAT在AMPH作用中的作用， 适当的生理和结构背景下，在以下具体目标：目的1。阐明的作用 膜相互作用在调节DAT的N末端磷酸化及其介导 AMPH诱导的DA外排和行为。AIM 2.为了确定N端磷酸化如何改变DAT 功能和动态。AIM 3.为了确定VMAT及其假定的N-末端磷酸化在 AMPH诱导的DA从突触囊泡在体内和体外流出。这项工作将提供一个明确的验证， 通过不改变DA摄取的机制阻断AMPH作用的药物的新靶点。

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.