Functional dynamics of glutamate transporters probed by high-speed atomic force microscopy with micro- to millisecond time resolution

通过微秒至毫秒时间分辨率的高速原子力显微镜探测谷氨酸转运蛋白的功能动力学

• 批准号: 10023957
• 负责人: Simon Scheuring
• 金额: $ 35.12万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10023957
• 关键词: Alzheimer' s Disease; Amino Acid Transporter; Amyotrophic Lateral Sclerosis; Archaea; Aspartate; Atomic Force Microscopy; Biological Models; Cells; Cognition; Coupled; Data; Dementia; Dependence; Deuterium; Development; Disease; Elevator; Epilepsy; Equilibrium; Excision; Excitatory Amino Acids; Family; Fluorescence Resonance Energy Transfer; Frequencies; Glutamate Receptor; Glutamate Transporter; Glutamates; Goals; Height; Homologous Gene; Human; Huntington Disease; Hydrogen; Image; Impairment; Individual; Integral Membrane Protein; Ion Channel; Kinetics; Ligands; Malignant Glioma; Measurement; Measures; Membrane; Membrane Lipids; Membrane Transport Proteins; Memory; Mental disorders; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Movement; Neuraxis; Neurodegenerative Disorders; Neurologic Process; Neurons; Neurotransmitters; Outcome; Physiological; Probability; Proteins; Pyrococcus horikoshii; Recycling; Reporting; Resolution; Role; Scanning; Schizophrenia; Sodium; Spectrum Analysis; Speed; Stroke; Structure; Synapses; Synaptic Cleft; Synaptic Transmission; System; Techniques; Technology; Temperature; Thermodynamics; Time; Toxic effect; Transmembrane Domain; Vertebrates; Vesicle; Visit; Work; base; biophysical techniques; experimental study; inhibitor/antagonist; insight; member; microscopic imaging; millisecond; movie; nervous system disorder; neuron loss; neurotoxic; neurotransmission; novel; novel strategies; postsynaptic; postsynaptic neurons; presynaptic; presynaptic neurons; prevent; reconstitution; single molecule; single-molecule FRET; solute; structured data; symporter; temporal measurement; thermophilic organism; tool; uptake

Glutamate or excitatory amino acid transporters (EAATs) are members of the Solute Carrier 1 family of transmembrane proteins. EAATs are key in the function of neuronal synapses responsible for the removal of excitatory neurotransmitters from the synaptic cleft after each neurotransmission. Malfunction of EAATs is involved in cerebral stroke, epilepsy, Alzheimer's disease, dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS) and malignant glioma. Thus, a profound understanding of the molecular determinants of EAAT function is crucial to understand these diseases. In the last decade, the structure and function of a prokaryotic glutamate transporter homolog, the sodium/aspartate symporter from archaebacterium Pyrococcus horikoshii, GltPh, has been extensively studied and made a perfect model system for EAAT studies. More recently a first structure of the human EAAT1 has been solved, but little is known about its transport dynamics and kinetics. In this project we will study both GltPh and hEAAT1 proteins, with the aim to characterize the so far elusive transport sub-states and kinetics in the ms and µs range. While the transport kinetics of the prokaryotic GltPh are slow (seconds to tens of milliseconds), EAATs are expected to work at faster rates (~100 to ~1000 transport cycles per second). Here, we will reconstitute GltPh and hEAAT1 in lipid membranes and employ high-speed atomic force microscopy (HS-AFM) to directly image transport cycles of individual unlabeled glutamate transporters with the aim to resolve transport-related conformational sub-states and fast kinetics. To achieve this goal, in addition to HS-AFM (HS-AFM) imaging, we develop and employ HS-AFM line scanning (HS-AFM-LS) and HS-AFM height spectroscopy (HS-AFM-HS). In these two novel sub-modes, we reach millisecond and microsecond time resolution, respectively. This makes our approach unique in three ways: i) We analyze the dynamics of membrane transporters in membrane. ii) We analyze unlabeled single transporter molecules. iii) We reach so far inaccessible temporal resolution. These novel approaches allow to unveil the occluded state in GltPh (only the outward and inward facing states in the transport cycle were assigned to date), its lifetime, the frequency with which it is being visited, and if it is visited on passage of a complete cycle or if returns from the occluded state occur regularly. In addition, given the fast transport rates of the eukaryotic homologues, we will pioneer single molecule dynamics measurements on human EAAT1 and provide first insights into their transport state probabilities and kinetics. The overall goal of the project is to establish a new experimental tool to study millisecond and microsecond dynamics in transporters and to apply this tool towards uncovering intermediates states in the transport cycles and measure currently inaccessible fast transport kinetics on the single molecule level.

谷氨酸或兴奋性氨基酸转运蛋白 (EAAT) 是溶质载体 1 家族的成员 跨膜蛋白。 EAAT 是神经元突触功能的关键，负责消除 每次神经传递后来自突触间隙的兴奋性神经递质。 EAAT 的故障是 涉及脑中风、癫痫、阿尔茨海默病、痴呆、亨廷顿病、肌萎缩侧索硬化症（ALS）和恶性神经胶质瘤。因此，对 EAAT 分子决定因素的深刻理解 功能对于了解这些疾病至关重要。近十年来，原核生物的结构和功能 谷氨酸转运蛋白同系物，来自古细菌火球菌 horikoshii 的钠/天冬氨酸同向转运蛋白， GltPh，已被广泛研究并为 EAAT 研究制定了完美的模型系统。最近第一次 人类 EAAT1 的结构已被解析，但对其运输动力学和动力学知之甚少。在 在这个项目中，我们将研究 GltPh 和 hEAAT1 蛋白，目的是表征迄今为止难以捉摸的蛋白 ms 和 µs 范围内的传输子状态和动力学。而原核 GltPh 的运输动力学 很慢（几秒到几十毫秒），EAAT 预计会以更快的速度工作（~100 到~1000 每秒传输周期）。在这里，我们将在脂质膜中重建 GltPh 和 hEAAT1，并使用 高速原子力显微镜（HS-AFM）可直接对单个未标记的传输周期进行成像 谷氨酸转运蛋白，旨在解决与转运相关的构象亚状态和快速动力学问题。到 为了实现这一目标，除了HS-AFM（HS-AFM）成像之外，我们还开发并采用了HS-AFM线扫描 (HS-AFM-LS) 和 HS-AFM 高度谱 (HS-AFM-HS)。在这两种新颖的子模式中，我们达到了 分别为毫秒和微秒时间分辨率。这使得我们的方法在三个方面独一无二： i）我们分析膜中膜转运蛋白的动力学。 ii) 我们分析未标记的单个 转运分子。 iii）我们达到了迄今为​​止无法达到的时间分辨率。这些新颖的方法允许 揭示 GltPh 中的封闭状态（仅传输周期中的向外和向内状态） 分配给日期），它的生命周期，它被访问的频率，以及它是否在通过时被访问 完整的循环或是否定期从闭塞状态返回。此外，考虑到快速的运输速度 真核同源物，我们将率先对人类 EAAT1 进行单分子动力学测量 并提供对其传输状态概率和动力学的初步见解。该项目的总体目标是 建立一种新的实验工具来研究转运蛋白中的毫秒和微秒动力学 应用此工具来揭示运输周期中的中间状态并测量当前 单分子水平上难以实现的快速传输动力学。