Structural Dynamics of the Human Serotonin Transporter

项目摘要 作为神经递质类的一员：钠协同转运体（NSSs）、血清素转运体（SERT） 通过再摄取或前向运输调节大脑中血清素的水平。SERT功能障碍 与人类的神经精神障碍和自闭症有关。因此，SERT是一个主要目标， 治疗发展以及滥用药物的作用部位。5-羟色胺从突触的转运 进入突触前细胞的神经元与钠离子和氯离子的同向转运在能量上耦合。但在 安非他明衍生物的存在下，这种机制被改变。安非他明类分子会诱导 通过NSSs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端介导的，N端是蛋白质-蛋白质转运的枢纽。 相互作用和信号传导，并由磷脂酰肌醇-4，5-二磷酸（PIP 2）调节，PIP 2是一种富含 神经元的质膜。该建议的中心目标是检查构象 动力学基础运输人类SERT（hSERT），阐明其N-末端的作用， 正向和反向运输。目标1将使用双电子-电子共振（DEER）光谱， 探针全长hSERT在不同条件下的构象平衡，以基准结构 在运输周期中的过渡。目标2将通过以下方法表征N-末端的构象： 调查的流动性和溶剂可及性的单一标记的网站，并确定其结构重组， PIP 2的存在。假设N-末端和PIP 2对hSERT施加结构变化 跨膜结构域，其在安非他明存在下促进反向转运。拟议 这些实验有望揭示真核细胞NSSs的转运机制， 这是理解疾病相关突变或外源分子（如 安非他明可扰乱这一机制。 Hassane Mchaourab博士的实验室专门揭示了 转运蛋白，包括NSS的细菌同系物，利用电子顺磁共振（EPR）工具 我们的合作者Eric Gouaux博士在真核NSS蛋白领域具有专业知识，并已确定 hSERT的几种高分辨率结构。Mchaourab博士的指导和 博士Gouaux将使我的目标中概述的实验成功，从哺乳动物蛋白质表达和 纯化通过EPR数据采集和分析，并最终在机械模型，集成高- 解析结构与构象动力学。

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.