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

谷氨酸或兴奋性氨基酸转运蛋白（EAAT）是谷氨酸转运蛋白1家族的成员。 跨膜蛋白EAAT是神经元突触功能的关键，神经元突触负责清除 兴奋性神经递质从突触间隙后的每一个神经传递。EAAT的故障是 涉及脑卒中、癫痫、阿尔茨海默病、痴呆、亨廷顿病、肌萎缩侧索硬化症（ALS）和恶性胶质瘤。因此，深刻理解EAAT的分子决定因素， 功能对于理解这些疾病至关重要。在过去的十年里，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Pyrococcus horikoshii的钠/天冬氨酸同向转运蛋白， GltPh已被广泛研究，并为EAAT研究提供了一个完美的模型系统。最近，第一次 人EAAT 1的结构已被解决，但对其转运动力学和动力学知之甚少。在 这个项目，我们将研究GltPh和hEAAT 1蛋白，目的是表征迄今为止难以捉摸的 在ms和µs范围内的传输子状态和动力学。而原核GltPh的转运动力学 速度较慢（秒至数十毫秒），EAAT预计将以更快的速度工作（约100至约1000 每秒传输周期）。在这里，我们将在脂质膜中重建GltPh和hEAAT 1，并使用 高速原子力显微镜（HS-AFM）直接成像单个未标记的运输循环 谷氨酸转运蛋白，目的是解决运输相关的构象亚状态和快速动力学。到 为了实现这一目标，除了HS-AFM（HS-AFM）成像，我们开发并采用HS-AFM线扫描 （HS-AFM-LS）和HS-AFM高度光谱（HS-AFM-HS）。在这两个新的子模式中，我们达到了 毫秒和微秒的时间分辨率。这使得我们的方法在三个方面独一无二： i）我们分析了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3.我们达到了迄今为止无法达到的时间分辨率。这些新方法允许 揭示了GltPh中的闭塞状态（仅在运输循环中向外和向内的状态被 它的寿命，它被访问的频率，如果它被访问通过一个 完整的循环或从阻塞状态返回是否定期发生。此外，考虑到 真核同源物，我们将开拓单分子动力学测量人类EAAT 1 并提供了对它们的输运状态概率和动力学的第一次洞察。该项目的总体目标是 建立一个新的实验工具，研究运输机的毫秒和微秒动力学， 应用这一工具来揭示运输周期中的中间状态，并测量当前 在单分子水平上不可接近的快速传输动力学。

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.