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

苯丙胺(Amphs)是一种被广泛使用和滥用的强效精神刺激剂，具有深远的医学意义 和社会影响。已知它们能使胞浆中的多巴胺(DA)动员到细胞外部 通过DA转运体(DAT)介导的外流，但调节这些作用的机制仍然很差 定义并是本提案的一个重点。利用异源表达系统和果蝇行为 模型，我们已经证明AMPH诱导的DA外流和随后的行为，但不是DA摄取，是 依赖于DAT的N端磷酸化。我们的团队还在理解 以细菌转运蛋白Leut为原型研究底物摄取的分子机制 最先进的单分子方法和计算分析。尽管N-端子区 我们的团队报告说，Leut基因基本上不存在，而在果蝇DAT(DDAT)结构中被截断 从从头算结构预测人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端到 高度动态化，含有二级结构元素，并通过 静电相互作用。在这里，我们的目标是探索这些结构元素，以深入了解 DAT及其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，我们的目标是探索调节amph诱导释放的机制。 DA从突触小泡进入细胞质。使用活的果蝇大脑的多光子成像技术 表明在药理上相关的浓度下，AMPHs必须被DAT主动转运 以及通过囊泡单胺转运体VMAT来减小囊泡pH梯度和 重新分配水泡内容物。尽管如此，这些事件是如何导致DA重新分布到细胞质的仍然存在 未知。最近的数据表明，VMAT N端的磷酸化在AMPH诱导的DA外流中是必不可少的 我们建议从机制上探索这一假说，并在体内进行测试。我们老牌的 多尺度方法结合了纯化蛋白质的生物化学和生物物理、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， 以及在活体苍蝇上的行为学研究，以探讨DAT和VMAT在AMPHs在 适当的生理和结构背景，在以下具体目标中：目标1.阐明 调节DAT N末端磷酸化的膜相互作用及其调节能力 AMPH诱导的DA外流和行为。目的2.确定N-末端磷酸化如何改变DAT 功能和动力学。目的3.确定VMAT及其N末端的磷酸化作用。 AMPH在体内和体外诱导突触小泡DA外流。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.