Structural Basis of Allosteric Regulation in the NMDA Receptor

NMDA 受体变构调节的结构基础

• 批准号: 9194311
• 负责人: MICHAEL Casey REGAN
• 金额: $ 5.92万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9194311
• 关键词: Achievement; Acids; Address; Affect; Agonist; Allosteric Regulation; Alzheimer' s Disease; Amino Acids; Binding; Binding Proteins; Binding Sites; Biochemical; Biophysics; Brain; Calcium; Calcium Channel; Calorimetry; Cell Death; Chemicals; Complex; Crystallization; Crystallography; Data; Development; Disease; Distant; Electrophysiology (science); Environment; Equipment; Event; Exons; Extracellular Domain; Floods; Functional disorder; Glutamate Receptor; Glutamates; Glycine; Goals; Hyperactive behavior; Immunoglobulin Fragments; Ion Channel; Ion Channel Protein; Kainic Acid Receptors; Knowledge; Laboratories; Learning; Ligand Binding; Ligand Binding Domain; Ligands; Link; Lipids; Long-Term Potentiation; Magnesium; Membrane Proteins; Memory; Mental Depression; Methods; Modernization; Molecular Conformation; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Neurons; Neuropharmacology; Neuroprotective Agents; Pharmacology; Phase; Phosphorylation; Polyamines; Proteins; RNA Splicing; Regulation; Research; Resolution; Roentgen Rays; Schizophrenia; Series; Side; Site; Spermine; Stroke; Structure; Synaptic Transmission; System; Techniques; Therapeutic; Titrations; Training; Translating; Transmembrane Domain; Validation; Variant; Work; Zinc; biophysical techniques; delta opioid receptor; experience; ifenprodil; insight; nervous system disorder; novel; protein protein interaction; public health relevance; receptor; small molecule; structural biology; targeted treatment; trait; voltage clamp

 DESCRIPTION (provided by applicant): The majority of synaptic transmission in the brain relies upon the ionotropic glutamate receptors. This class of ion channels is comprised of four subfamilies, and of these, the N-methyl-D-aspartate receptors (NMDARs) are critical for long-term potentiation and learning and their misregulation has been implicated in Alzheimer's disease, stroke, and a number of neurological disorders. The NMDAR assembles as an obligate heterotetramer with each subunit consisting of four modular domains: an extracellular Amino Terminal Domain (ATD) and Ligand Binding Domain (LBD), a transmembrane domain (TMD) which forms the ion channel, and an unstructured intracellular Carboxyl Terminal Domain (CTD). Numerous factors influence NMDAR activity, including pH, zinc & magnesium binding, and protein-protein interactions, at sites widely distributed across the protein, yet how these interactions are translated into differential channel activity remains largely unknown. This projec seeks to define the structural basis for the allosteric regulation of NMDAR activity through X-ray crystallographic, biophysical, and biochemical methods with two primary aims: 1) first, I will solve the X-ray crystal structure of the NMDAR ATD bound to a series of small molecules homologous to the antagonist Ifenprodil, which has considerable off-target effects but novel variations of which have been shown to bind the NMDAR only in the low-pH environments found during ischemic events such as stroke. 2) In my second aim, I will crystallize the intact receptor containing the ATD, LBD, and TMD in complex with the agonist spermine. Interestingly, this compound appears to bind at the ATD/LBD interface and specifically potentiates a subset of NMDAR splice variants; by solving and comparing the structures of the spermine-sensitive and spermine- insensitive receptors, I will determine how ligand binding to the extracellular domains allosterically alters the activity of the TMD. The Furukawa laboratory has extensive experience with the NMDAR, and I am uniquely situated to answer fundamental questions regarding NMDA receptor regulation through the use of our optimized mammalian expression system, advanced macromolecular crystallization techniques including lipidic cubic phase methods, and calorimetric and electrophysiological validation equipment. This research will allow me to gain tremendous experience in multiple expression systems and modern methods in electrophysiology and the structural biology of large membrane protein assemblies, and by defining the allosteric regulation of the NMDAR, my work has the potential to pave the way for the development of novel, highly-targeted therapeutics in a range of neurological disorders and diseases. Together, the training and the scientific achievements of this project will prepare me for research independence in this field.

 描述（由申请人提供）：大脑中的大部分突触传递依赖于离子型谷氨酸受体，此类离子通道由四个亚族组成，其中 N-甲基-D-天冬氨酸受体（NMDAR）是。 NMDAR 对长期增强和学习至关重要，其失调与阿尔茨海默病、中风和许多神经系统疾病有关。专性异四聚体，每个亚基由四个模块结构域组成：细胞外氨基末端结构域（ATD）和配体结合结构域（LBD）、形成离子通道的跨膜结构域（TMD）和非结构化细胞内羧基末端结构域（CTD）。许多因素影响 NMDAR 活性，包括 pH、锌和镁结合以及蛋白质与蛋白质之间的相互作用，这些相互作用广泛分布在蛋白质中，但这些相互作用如何转化为差异该项目旨在通过 X 射线晶体学、生物物理和生化方法来定义 NMDAR 活性变构调节的结构基础，主要目标有两个：1）首先，我将解决 X 射线晶体结构。 NMDAR ATD 与一系列与拮抗剂艾芬地尔同源的小分子结合，艾芬地尔具有相当大的脱靶效应，但其新变体已被证明仅在低 pH 环境中结合 NMDAR 2) 在我的第二个目标中，我将结晶包含 ATD、LBD 和 TMD 的完整受体，并与激动剂精胺复合，暗示该化合物似乎与 ATD/LBD 界面结合并特异性增强。 NMDAR 剪接变体的一个子集；通过求解和比较精胺敏感和精胺不敏感受体的结构，我将确定配体与细胞外结构域的结合如何变构地改变TMD。古河实验室在 NMDAR 方面拥有丰富的经验，我具有独特的优势，可以通过使用我们优化的哺乳动物表达系统、先进的大分子结晶技术（包括脂质立方相方法）以及量热和电生理学验证来回答有关 NMDA 受体调节的基本问题。这项研究将使我在电生理学和大型膜蛋白组件的结构生物学的多种表达系统和现代方法方面获得丰富的经验，并通过定义 NMDAR 的变构调节，我的工作有可能为一系列神经系统疾病和疾病的新型、高度针对性的治疗方法的开发铺平道路，该项目的培训和科学成就将为我在该领域的独立研究做好准备。