Structural characterization and targeting of tri-heteromeric NMDA receptors involved in neuroplasticity

参与神经可塑性的三异聚 NMDA 受体的结构表征和靶向

• 批准号: 10428597
• 负责人: Kevin Michalski
• 金额: $ 6.98万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428597
• 关键词: Address; Adopted; Affinity; Antibodies; Biochemistry; Biophysics; Brain; Cells; Chemicals; Cognition; Communication; Complex; Cryoelectron Microscopy; Development; Disease; Electrophysiology (science); Engineering; Excitatory Synapse; Exposure to; Family; Fellowship; Glutamate Receptor; Goals; Human; Immunotherapy; Insecta; Ion Channel; Laboratories; Lead; Learning; Link; Long-Term Depression; Long-Term Potentiation; Mediator of activation protein; Membrane; Membrane Proteins; Memory; Memory Loss; Mental Depression; Mental Health; Mental disorders; Modernization; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Neurologic; Neurologic Process; Neuronal Plasticity; Neurons; Neuropharmacology; Neurosciences; Pharmacology; Population; Positioning Attribute; Process; Production; Property; Prosencephalon; Protein Biochemistry; Proteins; Reagent; Research; Research Personnel; Research Training; Role; Schizophrenia; Scientist; Secure; Seizures; Signal Transduction; Site; Structure; Substance Addiction; Synapses; Synaptic plasticity; System; Techniques; Testing; Therapeutic; Training; Training Support; Work; Xenopus laevis; Yeasts; addiction; career; career development; cognitive capacity; experience; improved; memory retention; nanobodies; neurotransmission; neurotransmitter release; novel; particle; receptor; receptor expression; relating to nervous system; research and development; skills; structural biology; therapeutic target; three dimensional structure; tool; voltage

Abstract The goal of this proposal is to secure three years of postdoctoral research training support in order to structurally define in detail both GluN1-2A-2B and GluN1-2B-3A N-methyl-D-aspartate receptors (NMDARs) and to identify modulators that will specifically target these tri-heteromeric receptors without targeting di- heteromeric receptors. Reagents which specifically recognize and modulate the activity of GluN1-2A-2B and GluN1-2B-3A have the potential to boost cognition, and may lead to promising therapeutics to treat mental health disorders including depression, memory loss, and addiction. I have experience in electrophysiology with a background in ion channels and membrane protein biochemistry, and the proposed research and career development activities that I plan to pursue during this fellowship will provide the advanced training necessary to achieve my long-term career objective to be an independent researcher in the field of neuropharmacology. NMDARs belong to a large family of ionotropic glutamate receptors which are the prevailing regulators of neural communication, and their misregulation has been linked to numerous mental health disorders. Within the glutamate receptor family, heterotetrameric NMDARs are integral mediators of electrical and chemical signaling by sensing neurotransmitter release and transmembrane voltage potential at sites of neurotransmission known as synapses. Repeated activation of NMDARs results in synaptic plasticity in the forms of long term potentiation (LTP) and long term depression (LTD), which directly influence brain development and function including learning and memory. Changes in LTP and LTD are correlated with the specific activation of two NMDAR subtypes, the tri-heteromeric GluN1-2A-2B and GluN1-2B-3A receptors. However, how the activation of GluN1-2A-2B or GluN1-2B-3A correlates with changes in synaptic plasticity has been difficult to discern due to limited structural characterization of tri-heteromeric NMDARs and due to overlapping pharmacology profiles of these receptors with di-heteromeric NMDARs. In Aim 1, I will determine the structures of the human orthologues of GluN1-2A-2B and GluN1-2B-3A by cryo-electron microscopy, and in Aim 2 I will identify and test nanobody modulators that specifically target tri-heteromeric NMDARs using yeast display. The Furukawa Lab at Cold Spring Harbor Laboratory (CSHL) has vast experience working with NMDARs and is uniquely positioned to support my proposed aims using optimized NMDAR expression strategies, electrophysiology setups, and structural biology expertise. Additionally, I will have immediate access to a state-of-the-art cryo-EM facility as well as an on-campus Antibody and Display facility, which are both managed by highly regarded technical staff. The research proposed here will allow me to gain tremendous experience in modern structural biology techniques, display strategies used to develop novel immunotherapies, and has the potential to uncover subtype-specific NMDAR modulators that can address the effects of debilitating mental health disorders that I wish to pursue as an independent investigator.

抽象的 该建议的目的是确保三年的博士后研究培训支持，以便 结构上详细定义了Glun1-2a-2b和Glun1-2b-3a N-甲基-D-天冬氨酸受体（NMDARS） 并确定将专门针对这些三型杂体受体的调节剂，而无需针对DI- 杂体受体。特别识别和调节Glun1-2a-2b和 GLUN1-2B-3A有可能提高认知，并可能导致有前途的治疗剂治疗心理 健康障碍，包括抑郁症，记忆力丧失和成瘾。我有电生理学的经验 离子渠道和膜蛋白生物化学的背景以及拟议的研究和职业 我计划在此奖学金期间进行的开发活动将提供必要的高级培训 为了实现我的长期职业目标，成为神经药理学领域的独立研究人员。 NMDAR属于一个大型的离子型谷氨酸受体，这是主要的调节剂 神经沟通及其不利地位与多种心理健康障碍有关。之内 谷氨酸受体家族，杂元NMDAR是电气和化学的积分介体 通过感测神经递质释放和跨膜电压的信号传导 神经传递称为突触。反复激活NMDAR会导致突触可塑性 长期增强（LTP）和长期抑郁（LTD）的形式，直接影响大脑 发展和功能，包括学习和记忆。 LTP和LTD的变化与 两种NMDAR亚型的特异性激活，即三源体GLUN1-2A-2B和GLUN1-2B-3A受体。 但是，GLUN1-2A-2B或GLUN1-2B-3A的激活与突触可塑性的变化如何相关 由于三盐分内NMDAR的结构表征有限，因此很难辨别 这些受体与二型型NMDAR的重叠药理学概况。在AIM 1中，我将确定 glun1-2a-2b和Glun1-2b-3a的人类直系同源物的结构，通过冷冻电子显微镜，以及 在AIM 2中，我将识别和测试使用专门针对三型直晶NMDAR的纳米机构调节剂 酵母显示。冷春港实验室（CSHL）的Furukawa实验室拥有丰富的经验 NMDAR，并具有独特的位置，可以使用优化的NMDAR表达式支持我的拟议目标 策略，电生理设置和结构生物学专业知识。此外，我将立即 使用最先进的冷冻EM设施以及校园抗体和展示设施 两者都由备受推崇的技术人员管理。这里提出的研究将使我获得 现代结构生物学技术的巨大经验，用于发展新颖的策略 免疫疗法，并且有可能发现可以解决该问题的亚型特异性NMDAR调节剂 我希望作为独立研究者追求的精神健康障碍的影响。

项目成果

Structural insights into binding of therapeutic channel blockers in NMDA receptors.

• DOI: 10.1038/s41594-022-00772-0
• 发表时间: 2022-06
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: Chou, Tsung-Han;Epstein, Max;Michalski, Kevin;Fine, Eve;Biggin, Philip C.;Furukawa, Hiro
• 通讯作者: Furukawa, Hiro

On the molecular nature of large-pore channels.

• DOI: 10.1016/j.jmb.2021.166994
• 发表时间: 2021-08-20
• 期刊: Journal of molecular biology
• 影响因子: 5.6
• 作者: Syrjanen J;Michalski K;Kawate T;Furukawa H
• 通讯作者: Furukawa H

Effective production of oligomeric membrane proteins by EarlyBac-insect cell system.

• DOI: 10.1016/bs.mie.2020.12.019
• 发表时间: 2021
• 期刊: Methods in enzymology
• 作者: Furukawa H;Simorowski N;Michalski K
• 通讯作者: Michalski K