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: 10471928
• 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/10471928
• 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; 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; 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（EAATS）是溶质载体1家族的成员 跨膜蛋白。 EAAT是负责去除的神经元突触功能的关键 每种神经传递后突触裂口的兴奋性神经递质。 Eaats的故障是 包括在脑卒中，癫痫，阿尔茨海默氏病，痴呆症，亨廷顿氏病，肌萎缩性侧面硬化症（ALS）和恶性神经胶质瘤中。这是对EAAT的分子决定者的深刻理解 功能对于了解这些疾病至关重要。在过去的十年中，核的结构和功能 谷氨酸转运蛋白同源物，来自考古杆菌horikoshii的钠/天冬氨酸对甲酸酯的分类剂， GLTPH已被广泛研究，并为EAAT研究建立了完美的模型系统。最近是第一个 人类EAAT1的结构已经解决，但对其运输动力学和动力学知之甚少。在 这个项目我们将研究GLTPH和HEAAT1蛋白，目的是表征到目前为止难以捉摸的 在MS和µS范围内的传输子状态和动力学。而原核生物的传输动力学 慢速（秒至数十毫秒），EAAT的工作速度更快（〜100至〜1000） 每秒运输周期）。在这里，我们将在脂质膜中重新建立GLTPH和HEAAT1并使用 高速原子力显微镜（HS-AFM）直接成像单个未标记的传输周期 谷氨酸转运蛋白的目的是解决与运输相关的构象子态和快速动力学。到 实现此目标，除了HS-AFM（HS-AFM）成像外，我们还开发和采用HS-AFM线扫描 （HS-AFM-LS）和HS-AFM高度光谱（HS-AFM-HS）。在这两个新颖的子模型中，我们到达 分别毫秒和微秒的时间分辨率。这使我们的方法以三种方式与众不同： i）我们分析膜中膜转运蛋白的动力学。 ii）我们分析了未标记的单个 转运蛋白分子。 iii）我们到目前为止无法访问临时分辨率。这些新颖的方法允许 揭示GLTPH中的封闭状态（在运输周期中只有向外和向内的状态 分配到迄今为止），其寿命，访问频率的频率，以及是否在通过时访问 完整的周期或是否从封闭状态进行返回。此外，考虑到快速运输速率 真核同源物，我们将先驱对人EAAT1的单分子动力学测量 并为其运输状态的可能性和动力学提供初次见解。该项目的总体目标是 建立一个新的实验工具，以研究转运蛋白和转运蛋白的微秒和微秒动力学 将此工具应用于运输周期中的中间体状态并目前 单分子水平上无法接近的快速传输动力学。