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

摘要 这项提议的目标是确保三年的博士后研究培训支持，以便 在结构上详细定义GluN1-2A-2B和GluN1-2B-3A N-甲基-D-天冬氨酸受体(NMDAR) 并确定将专门靶向这些三异构体受体的调节剂，而不是靶向二聚体。 异构体受体。特异性识别和调节GluN1-2A-2B活性的试剂和 GluN1-2B-3A具有提高认知能力的潜力，并可能导致治疗精神障碍的有前途的疗法 健康障碍，包括抑郁、记忆力减退和成瘾。我在电生理学方面有经验 离子通道和膜蛋白生物化学的背景，以及建议的研究和职业 我计划在这次奖学金期间从事的发展活动将提供必要的高级培训 为了实现我长期的职业目标，成为神经药理学领域的一名独立研究员。 NMDAR属于一个离子亲和性谷氨酸受体大家族，它是 神经通讯，以及它们的错误调节与许多精神健康障碍有关。在 谷氨酸受体家族，异四聚体NMDAR是电和化学的综合介质 感觉神经递质释放和跨膜电压电位的信号传导 神经传递称为突触。NMDAR的反复激活导致大鼠脑内突触的可塑性 直接影响大脑的长时程增强(LTP)和长时程增强(LTD)的形式 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，三异构体GluN1-2A-2B和GluN1-2B-3A受体。 然而，GluN1-2A-2B或GluN1-2B-3A的激活如何与突触可塑性的变化相关 由于三异构体NMDAR的有限结构特征以及由于 这些受体与双异构体NMDAR的药理图谱重叠。在目标1中，我将确定 用冷冻电子显微镜观察GluN1-2A-2B和GluN1-2B-3A的人类同源物的结构，以及 在目标2中，我将识别和测试专门针对三异构体NMDAR的纳米体调节剂 酵母展示。冷泉港实验室(CSHL)的古川实验室拥有丰富的工作经验 NMDAR，其独特定位是使用优化的NMDAR表达式支持我建议的目标 策略、电生理学设置和结构生物学专业知识。另外，我会马上要一份 进入最先进的冷冻-EM设施以及校园抗体和展示设施，这些设施是 这两家公司都由备受尊敬的技术人员管理。这里提出的研究将使我获得 在现代结构生物学技术、展示策略方面具有丰富的经验，用于开发新奇 免疫疗法，并有可能发现亚型特异性NMDAR调节剂，可以解决 作为一名独立调查员，我希望研究衰弱的精神健康障碍的影响。

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.