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（由适用提供）：大脑中的大多数突触传播依赖于离子型谷氨酸受体。这类离子通道由四个亚家族组成，其中N-甲基-D-天冬氨酸受体（NMDARS）对于长期增强和学习至关重要，并且在阿尔茨海默氏病，中风，许多神经系统疾病中实施了错误调节。 NMDAR作为强制性异驱动器组装，每个亚基由四个模块化结构域组成：细胞外氨基末端结构域（ATD）和配体结合结构域（LBD），一个跨膜结构域（TMD），形成离子通道，以及一个非结构性的细胞内箱内碳纤维式碳酰基端子域（CTD）。许多因素会影响NMDAR活性，包括pH，锌和镁结合以及蛋白质 - 蛋白质相互作用，在广泛分布在整个蛋白质的位置，但是如何将这些相互作用转化为差异通道活性仍然很大。 This project 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仅在诸如中风等缺血性事件中发现的低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的完整受体与激动剂精子中的复合物。有趣的是，该化合物似乎在ATD/LBD界面上结合，并特别具有NMDAR剪接变体的子集。通过求解和比较对精子敏感和对精子不敏感受体的结构，我将确定配体与细胞外结构域的结合如何变化，从而改变TMD的活性。 Furukawa实验室在NMDAR方面具有丰富的经验，我在使用我们优化的哺乳动物表达系统，晚期大分子分子结晶技术（包括脂质立方体方法）以及载脂情况和载脂物质和电子生理学验证的设备（包括脂质的高级分子结晶技术）来回答有关NMDA受体调节的基本问题。这项研究将使我能够在多种表达系统和电生理学的现代方法以及大膜蛋白质组件的结构生物学方面获得丰富的经验，并通过定义NMDAR的变构调节，我的工作有可能为新颖的，高度针对性的神经性疾病和疾病范围内的新型治疗方法铺平道路。该项目的培训和科学成就一起将使我为该领域的研究独立做好准备。