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: 10240704
• 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/10240704
• 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; 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