Structural Dynamics of the Human Serotonin Transporter

项目总结 作为神经递质的一员：钠转运体(NSSS)，5-羟色胺转运体(SERT) 通过重新摄取或前向转运来调节大脑中的5-羟色胺水平。SERT功能障碍一直以来 与人类的神经精神障碍和自闭症有关。因此，SERT是 滥用药物的治疗发展和作用部位。5-羟色胺从突触的运输 进入突触前细胞与钠离子和氯离子的结合是能量耦合的。然而，在 在苯丙胺衍生物的存在下，这一机制被改变。安非他明类分子能诱导 NSSS的反向转运，导致5-羟色胺、多巴胺和去甲肾上腺素的突触水平增加。 这种反向转运机制是由转运蛋白的N-末端介导的，转运蛋白是蛋白质-蛋白质的枢纽 相互作用和信号传递，并受磷脂酰肌醇-4，5-二磷酸(PIP2)调节，PIP2是一种富含 神经元的质膜。这项建议的中心目标是研究构象 人类SERT(HSERT)转运的动力学基础，阐明其N末端在两者中的作用 正向和反向传输。目标1将使用双电子-电子共振(DeER)光谱来 探索全长hSERT在不同条件下的构象平衡以作为结构基准 在整个运输周期中的过渡。目标2将通过以下方式表征N末端的构象 调查单一标记位点的流动性和溶剂可及性，并确定其结构重组 PIP2的存在。N末端和PIP2被假设为对hSERT施加结构变化 跨膜结构域，在苯丙胺存在时促使反向转运。建议数 实验有望揭示真核细胞NSSS的运输机制，并可能 对于理解与疾病相关的突变或外源分子(如 安非他命可能会扰乱这种机制。 Hassane Mchaourab博士的实验室专门研究 转运体，包括NSS的细菌同源物，利用电子顺磁共振(EPR)工具 我们的合作者Eric Gouaux博士在真核细胞NSS蛋白领域拥有专业知识，并已确定 HSERT的几种高分辨率结构。总而言之，Mchaourab博士的指导和 Gouaux博士将使我的目标中概述的实验从哺乳动物蛋白质表达和 通过EPR数据采集和分析进行提纯，并最终形成集成了高 具有构象动力学的分辨结构。

PROJECT SUMMARY As a member of the class of neurotransmitter:sodium symporters (NSSs), the serotonin transporter (SERT) regulates levels of serotonin in the brain through reuptake or forward transport. Dysfunction of SERT has been associated with neuropsychiatric disorders and autism in humans. As such, SERT is a major target for therapeutic development as well as sites of action of drugs of abuse. Transport of serotonin from the synapse into the presynaptic cell is energetically coupled to symport of sodium and chloride ions. However, in the presence of amphetamine derivatives, this mechanism is altered. Molecules in the amphetamine class induce reverse transport by NSSs, leading to increased synaptic levels of serotonin, dopamine, and norepinephrine. This reverse transport mechanism is mediated by the N-terminus of the transporter, a hub for protein-protein interactions and signaling, and is modulated by phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid enriched in the plasma membrane of neurons. The central objective of this proposal is to examine the conformational dynamics underlying transport for human SERT (hSERT), illuminating the role of its N-terminus in both forward and reverse transport. Aim 1 will use double electron-electron resonance (DEER) spectroscopy to probe conformational equilibria of full-length hSERT under different conditions to benchmark structural transitions across the transport cycle. Aim 2 will characterize the conformation of the N-terminus through surveying the mobility and solvent accessibility of singly labeled sites and define its structural reorganization in the presence of PIP2. The N-terminus and PIP2 are hypothesized to impose structural changes to the hSERT transmembrane domain, which prompts reverse transport in the presence of amphetamine. The proposed experiments are expected to reveal insights into the transport mechanism of eukaryotic NSSs and could be foundational for understanding how the presence of disease-linked mutations or exogenous molecules such as amphetamine could disturb this mechanism. The laboratory of Dr. Hassane Mchaourab specializes in revealing the conformational dynamics of transporters, including bacterial homologs of NSS, utilizing the tools of electron paramagnetic resonance (EPR) Our collaborator, Dr. Eric Gouaux has expertise in the field of eukaryotic NSS proteins and has determined several high-resolution structures of hSERT. Together, the mentorship of Dr. Mchaourab and the guidance of Dr. Gouaux will enable the success of experiments outlined in my aims from mammalian protein expression and purification through EPR data acquisition and analysis and culminating in mechanistic models that integrate high- resolution structures with conformational dynamics.