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

摘要 该提案的目标是确保三年的博士后研究培训支持，以 在结构上详细定义GluN 1 -2A-2B和GluN 1 -2B-3A N-甲基-D-天冬氨酸受体（NMDAR） 并鉴定将特异性靶向这些三异聚体受体而不靶向二异聚体受体的调节剂。 异聚受体特异性识别和调节GluN 1 -2A-2B活性的试剂， GluN 1 -2B-3A具有促进认知的潜力，并可能导致有希望的治疗精神疾病的方法。 健康障碍，包括抑郁症、记忆丧失和成瘾。我在电生理学方面有经验， 在离子通道和膜蛋白生物化学的背景，以及拟议的研究和职业生涯 我计划在此期间从事的开发活动将提供必要的高级培训 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属于离子型谷氨酸受体的大家族，其是NMDAR的主要调节剂。 神经交流及其失调与许多精神疾病有关。内 谷氨酸受体家族，异四聚体NMDAR是电和化学的整合介体， 通过感知神经递质释放和跨膜电压电位来进行信号传导， 也就是突触。NMDAR的重复激活导致突触可塑性， 长时程增强（LTP）和长时程抑制（LTD）的形式，直接影响大脑 发育和功能，包括学习和记忆。LTP和LTD的变化与 特异性激活两种NMDAR亚型，三异聚体GluN 1 -2A-2B和GluN 1 -2B-3A受体。 然而，GluN 1 -2A-2B或GluN 1 -2B-3A的激活如何与突触可塑性的变化相关， 由于三异聚体NMDAR的结构表征有限， 这些受体与二异聚体NMDAR的药理学特征重叠。在目标1中，我将确定 通过冷冻电子显微镜观察GluN 1 -2A-2B和GluN 1 -2B-3A的人类直向同源物的结构，以及 在目的2中，我将使用以下方法鉴定和测试特异性靶向三异源NMDAR的纳米抗体调节剂： 酵母展示冷泉港实验室（CSHL）的古川实验室在以下方面拥有丰富的经验： NMDAR和独特的定位，以支持我提出的目标，使用优化的NMDAR表达 策略、电生理学设置和结构生物学专业知识。另外，我会立即 访问最先进的冷冻EM设施以及校园内的抗体和显示设施，这是 都由德高望重的技术人员管理。这里提出的研究将使我获得 在现代结构生物学技术方面的丰富经验，用于开发新的 免疫疗法，并有可能揭示亚型特异性NMDAR调节剂，可以解决 我希望作为一名独立的调查者继续研究这种精神疾病的影响。

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.