Molecular Mechanisms, Modulation, and Synaptic Organization of Kainate Receptors

红藻氨酸受体的分子机制、调节和突触组织

• 批准号: 10417222
• 负责人: Joshua Levitz
• 金额: $ 42.38万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-03 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10417222
• 关键词: AMPA Receptors; Address; Area; Biochemical; Biology; Brain; Cations; Characteristics; Complex; Cryoelectron Microscopy; Data; Development; Drug Targeting; Electrophysiology (science); Epilepsy; Family; Fluorescence; Glutamate Receptor; Glutamates; Goals; Homo; Human; Ion Channel; Kainic Acid Receptors; Kinetics; Knowledge; Mediating; Molecular; Mood Disorders; N-Methyl-D-Aspartate Receptors; Nature; Neurotransmitters; Physiological; Physiology; Positioning Attribute; Preparation; Property; Proteins; Research; Resolution; Role; Shapes; Structure; Synapses; Synaptic Transmission; System; Work; desensitization; gamma-Aminobutyric Acid; insight; member; millisecond; neurotransmitter release; novel therapeutics; pain perception; programs; receptor; single molecule

Molecular Mechanisms, Modulation, and Synaptic Organization of Kainate Receptors Kainate receptors (KARs) are members of the ionotropic glutamate receptor (iGluR) family of cation channels, which also includes AMPA and NMDA receptors. They localize to synapses where they respond to L- glutamate (L-Glu) which is the most abundant excitatory neurotransmitter in the brain. KARs serve a canonical role in synaptic depolarization, and non-canonical roles in modulating L-Glu and GABA release and synapse maturation. They are involved in pain perception, epilepsy and mood disorders which has made them important drug targets. Unfortunately, our understanding of KAR molecular mechanisms is limited without structures for the most abundant KAR complexes in the brain. My research program aims to address this gap by developing molecular mechanisms for KAR gating, modulation, and organization. KARs assemble as tetramers from a pool of five subunits (GluK1-5). After assembly and positioning at the synapse, they respond to synaptic L-Glu by opening their ion channel and, under the sustained presence of L-Glu, desensitize within milliseconds to close their channel. A goal of my lab is to understand how KAR subunits are organized in the receptor, and how the subunits coordinate together to control the ion channel. My recent cryo-electron microscopy (cryo-EM) work revealed the surprising organization, symmetries, and domain interfaces of homo-tetrameric GluK2 (Meyerson et al. 2014 Nature; Meyerson et al. 2016 Nature). However, these results are of limited physiological value because native KARs in the brain are hetero-tetramers composed of GluK2 and GluK5 subunits. The GluK2/K5 hetero-tetramer has strict assembly rules and unique kinetic properties which underpin KAR physiology, but little is known of its subunit organization, or activation and desensitization mechanisms. Our preliminary data show successful expression, purification, and initial structure determination of the GluK2/K5 heteromer to 3.7 Å resolution with cryo-EM. In research Area 1 we aim to determine how GluK2/K5 organizes and functions by using a combination of single molecule fluorescence, electrophysiology, and cryo-EM. KARs do not function in isolation. Synaptic KAR complexes incorporate transmembrane Neto2 auxiliary proteins which shapes their characteristic kinetic profile. KARs also attach to C1ql2 proteins which are required for proper receptor localization. Understanding how KARs interact with these proteins is essential to bridging the gap between molecular mechanism and synapse biology and is a major research goal. Towards this end we have developed new biochemical preparations which enable detailed mechanistic interrogation on these systems. In Area 2 we will determine how KARs interact with and are modulated by C1ql2 and Neto2. I hypothesize that our work in research Areas 1 and 2 will give important insights into KAR physiology.

Kaiinate受体的分子机制，调制和突触组织 Kaiinate受体（KARS）是阳离子的离子型谷氨酸受体（Iglur）家族的成员 频道，其中还包括AMPA和NMDA接收器。他们本地化以对l-响应的突触 谷氨酸（L-GLU）是大脑中最丰富的兴奋性神经递质。 KARS为规范服务 在突触沉积中的作用，以及在调节L-GLU和GABA释放和突触中的非规范作用 成熟。他们参与了疼痛感，癫痫和情绪障碍，这使他们 重要的药物靶标。不幸的是，我们对KAR分子机制的理解是有限的 大脑中最丰富的KAR复合物的结构。我的研究计划旨在解决 通过开发用于KAR门控，调制和组织的分子机制，这一差距。 KARS作为四个亚基的四聚体（GLUK1-5）组装。组装和定位 突触，他们通过打开其离子通道对突触L-GLU做出反应，并在持续存在下 L-Glu在毫秒内脱敏以关闭其频道。我实验室的目标是了解KAR如何 亚基是在接收器中组织的，以及亚基如何坐标以控制离子通道。 我最近的低温电子显微镜（Cryo-EM）的工作揭示了令人惊讶的组织，对称性和 同型gluk2的域界面（Meyerson等人，2014年； Meyerson等人，2016年自然）。 但是，这些结果的物理价值有限，因为大脑中的天然KAR是异性四聚体 由gluk2和gluk5亚基组成。 Gluk2/K5异核四聚体具有严格的组装规则和独特的规则 基于KAR生理学的动力学特性，但对其亚基组织或激活知之甚少 和脱敏机制。我们的初步数据显示了成功的表达，纯化和初始数据 用冷冻EM分辨率将GLUK2/K5异构体的结构测定到3.7Å。在研究区域1 我们旨在通过使用单个组合来确定GLUK2/K5如何组织和功能 分子荧光，电生理学和冷冻EM。 KAR不隔离起作用。突触KAR复合物包含跨膜Neto2 辅助蛋白塑造其特征动力学谱。 KAR还附着在C1QL2蛋白上 适当的受体定位需要。了解KARS与这些蛋白质的相互作用是必不可少的 弥合分子机制和突触生物学之间的差距，并且是一个主要的研究目标。向 我们已经开发了新的生化制剂，可以对 这些系统。在区域2中，我们将确定KARS如何与C1QL2相互作用并调节 和Neto2。我假设我们在研究领域1和2中的工作将为KAR提供重要的见解 生理。