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Single-Particle Cryo-EM Characterization of AMPA Receptor Functional States

AMPA 受体功能状态的单粒子冷冻电镜表征

基本信息

批准号：
10654933
负责人：
Alexander Sobolevsky
金额：
$ 41.11万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10654933
关键词：
AMPA Receptors Address Agonist Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Architecture Area Binding Binding Sites Biological Assay Brain Cells Clinical Communication Complex Coupling Cryoelectron Microscopy Crystallography DLG1 gene Detection Drug Design Electrophysiology (science)Epilepsy Family Fluorescence Future Generations Genes Germ Cells Glutamate Receptor Glutamates Goals Homologous Gene Individual Ion Channel Ischemia Knowledge Length Ligand Binding Domain Mediating Membrane Molecular Molecular Conformation Molecular Sieve Chromatography Neurons Ozone Pathologic Pharmaceutical Preparations Primary Lateral Sclerosis Process Protein Biochemistry Protein Engineering Proteins Receptor Activation Regulation Reporting Research Resolution Rest Role Signal Transduction Site-Directed Mutagenesis Structural Models Structure Synapses Synaptic Receptors Synaptic Transmission Technology Testing Work X-Ray Crystallography antagonist cognitive function cognitive process desensitization design genetic regulatory protein glutamatergic signaling member nervous system development nervous system disorder neurotransmission novel therapeutics particle patch clamp positive allosteric modulator postsynaptic protein expression receptor receptor binding receptor function response small molecule structural determinants success thermostability trafficking transmission process

项目摘要

PROJECT SUMMARY AMPA-subtype ionotropic glutamate receptors mediate fast signaling between neurons and contribute to high cognitive processes. Since AMPA receptors are also implicated in numerous neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, epilepsy, and ischemia, the ability to regulate them represents an important clinical goal. However, there is an unmet need for drugs to regulate AMPA receptor activity in pathological conditions, which highlights a profound gap in our knowledge of AMPA receptor structure and function. Our long-term goal is to understand how AMPA receptor molecular machinery operates at the atomic level. We plan to study AMPA receptor structure and function using single-particle cryo-electron microscopy (cryo-EM). Advances in cryo-EM that were signified by the “resolution revolution” brought us a number of AMPA receptor structures in different gating conformations and in complex with several small molecules and regulatory proteins. Nevertheless, only a little fraction of AMPA receptor conformational states and binding partners have been characterized structurally and the resolution of the reported structures remains relatively low, limiting our ability to see the atomic details. We, therefore, plan to use cryo-EM advances to fill up this knowledge gap and to focus on the following Specific Aims: (1) unravel structural principles of AMPA receptor regulation by auxiliary subunits, (2) reveal molecular determinants of AMPA receptor activation and conductance, and (3) determine molecular mechanisms of AMPA receptor desensitization. To reach our research goals, we will use cryo-EM to obtain structures of AMPA receptors alone or in complex with different auxiliary proteins, in the presence of agonists, competitive antagonists, positive or negative allosteric modulators, and in conditions favoring various conformational states. We will use Fluorescence-detection Size Exclusion Chromatography (FSEC) and thermostability assays to assess protein expression, assembly, homogeneity, and stability. We will also employ site-directed mutagenesis combined with single-channel and whole-cell patch-clamp electrophysiological recordings to study functional mechanisms and to critically test our structural models. Reaching our research goals will have a significant impact on understanding the mechanisms of excitatory neurotransmission and will provide molecular-level knowledge essential to facilitate the design of new drugs for the treatment of neurological disorders.

项目总结