Structural Dynamics of the Human Serotonin Transporter

人类血清素转运蛋白的结构动力学

• 批准号: 10610326
• 负责人: Alexandra Corinne Schwartz
• 金额: $ 3.31万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10610326
• 关键词: 3-Dimensional; American; Amphetamine Addiction; Amphetamines; Benchmarking; Biochemical; Brain; Cell membrane; Cells; Chloride Ion; Collaborations; Coupled; Cryoelectron Microscopy; Crystallography; Data; Data Analyses; Dimensions; Disease; Dopamine; Drosophila melanogaster; Drug Design; Electron Spin Resonance Spectroscopy; Electrons; Elements; Engineering; Equilibrium; Fingerprint; Functional disorder; Future; Goals; Homologous Gene; Human; Ion Cotransport; Knowledge; Label; Laboratories; Length; Ligands; Light; Link; Lipid Binding; Lipids; Mediating; Membrane; Mental Depression; Mental disorders; Mentorship; Methodology; Modeling; Molecular; Molecular Conformation; Mutation; N-terminal; Neurons; Neurotransmitters; Norepinephrine; Phosphatidylinositol 4,5-Diphosphate; Physiological; Post-Translational Protein Processing; Presynaptic Terminals; Proteins; Research; Resolution; Role; Sampling; Serotonin; Signal Transduction; Site; Sodium; Solvents; Spectrum Analysis; Spin Labels; Stimulant; Structure; Surveys; Synapses; Testing; Transmembrane Domain; abuse liability; addiction; autism spectrum disorder; computer studies; conformational conversion; data acquisition; dopamine transporter; drug action; drug of abuse; ecstasy; experimental study; in vivo; insight; member; neuropsychiatric disorder; neurotransmitter transport; novel therapeutics; presynaptic; protein expression; protein protein interaction; protein purification; reuptake; serotonin transporter; skills; sodium ion; success; symporter; targeted treatment; therapeutic development; tool

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.

项目摘要 作为神经递质类别的成员：钠共肌（NSS），5-羟色胺转运蛋白（SERT） 通过再摄取或前进运输来调节大脑中5-羟色胺的水平。 Sert的功能障碍已经 与人类的神经精神疾病和自闭症有关。因此，Sert是 治疗发展以及滥用药物的作用部位。从突触中传输5-羟色胺 进入突触前细胞在能量耦合与钠和氯离子的同步耦合。但是，在 苯丙胺衍生物的存在，这种机制发生了变化。苯丙胺类中的分子诱导 NSS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升高。 这种反向传输机制是由转运蛋白的N末端介导的，蛋白质蛋白的枢纽 相互作用和信号传导，并由磷脂酰肌醇-4,5-双磷酸（PIP2）调节，一种富含脂质的脂质 神经元的质膜。该提议的核心目的是检查构象 人类Sert运输的基础动力（HSERT），阐明了其N末端在两者中的作用 向前和反向运输。 AIM 1将使用双电子 - 电子共振（DEER）光谱 全长HSERT在不同条件下的探针构象平衡至基准结构 整个运输周期的过渡。 AIM 2将通过 调查单一标记地点的移动性和溶剂可及性，并定义其结构重组 PIP2的存在。假设N末端和PIP2对HSERT施加结构性变化 跨膜结构域，在苯丙胺存在下促使反向转运。提议 预计实验会揭示对真核NSS的运输机制的见解，可能是 理解疾病连接突变或外源分子（例如）的基础 苯丙胺可能会干扰这种机制。 Hassane Mchaourab博士的实验室专门揭示 使用电子顺磁共振（EPR）的工具，包括NSS的细菌同源物（包括NSS的细菌同源物） 我们的合作者Eric Gouaux博士在真核NSS蛋白质领域具有专业知识，并确定了 HSERT的几个高分辨率结构。麦克劳布博士的指导和指导在一起 Gouaux博士将使我从哺乳动物蛋白表达和 通过EPR数据采集和分析纯化，并在整合高级的机理模型中进行纯化 具有构象动力学的分辨率结构。