Guanidinium Toxins as Molecular Probes for NaV Study

胍毒素作为 NaV 研究的分子探针

• 批准号: 10374137
• 负责人: Justin Du Bois
• 金额: $ 43.62万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10374137
• 关键词: Action Potentials; Acute; Address; Affect; Affinity; Affinity Chromatography; Antibodies; Arrhythmia; Binding; Binding Proteins; Biological Phenomena; Biotin; Cell Shape; Cell membrane; Cells; Cellular Membrane; Chemical Agents; Chemicals; Chemistry; Collection; Complex; Cryoelectron Microscopy; Cues; Cysteine; Development; Digit structure; Disease; Drug Kinetics; Electrophysiology (science); Engineering; Epilepsy; Epitopes; Fluorescent Dyes; Generations; Genetic Variation; Goals; Human; Human Pathology; Image; Individual; Inflammation Mediators; Inflammatory; Injury; Investigation; Ions; Kinetics; Label; Lead; Literature; Lysine; Measures; Medicine; Membrane; Membrane Proteins; Methods; Modeling; Modification; Molecular; Molecular Probes; Movement; Mutagenesis; Myopathy; Natural Products; Nature; Nerve Block; Neuroglia; Neurons; Oral cavity; Organism; Penetration; Physiology; Population; Post-Translational Protein Processing; Process; Property; Protein Isoforms; Proteins; Proteomics; Reagent; Reporting; Research; Role; Saxitoxin; Shapes; Signal Transduction; Site; Skeletal Muscle; Sodium Channel; Source; Structure; Study models; Surface; Tetrodotoxin; Therapeutic; Tissues; Toxin; Training; Vestibule; Work; alpha Toxin; base; bioelectricity; cell fixing; cell type; chemical synthesis; experimental study; extracellular; guanidinium; human disease; hydrophilicity; imaging agent; inhibitor; insight; interest; live cell imaging; mutant; nanomolar; nerve injury; nervous system disorder; neuronal excitability; neurotransmission; painful neuropathy; programs; protein complex; protein degradation; protein transport; response; small molecule; sodium ion; spatiotemporal; success; tool; voltage

PROJECT SUMMARY Neuronal excitability relies on the tightly regulated expression and discrete subcellular localization of voltage-gated sodium ion channels (NaVs). These large membrane protein complexes control the movement of sodium ions across cell membranes and are responsible for initiating and propagating action potentials. A desire to better understand the role of NaV subtypes in electrical signal conduction and the relationship between channel dysregulation and specific human pathologies (e.g., arrhythmia, epilepsy, skeletal muscle disorders, neuropathic pain) motivates the development of high precision reagents to facilitate NaV studies. Investigations of NaV physiology are currently limited by a lack of available methods with which to modulate the function of individual channel subtypes and to mark changes in cellular distributions, membrane expression levels, and structural modifications (i.e., protein post- translational modification) in live cells and in response to external cues. We are developing small molecule probes for NaV studies based on naturally occurring bis- guanidinium toxins, among which saxitoxin (STX) is the archetype. These agents function as molecular ‘corks’ to occlude the extracellular mouth of the ion conductance pore, a desirable feature for the types of tool compounds we wish to access. De novo chemical synthesis has enabled the engineering of modified forms of STX, which we will use in combination with protein mutagenesis and electrophysiology to investigate NaV activity on action potential dynamics. Understanding how NaV expression is regulated and altered by extrinsic factors—glial cells, inflammatory mediators, pH, nerve injury—and how such changes modulate action potentials is a long-term goal of our research. To address these questions, we will develop and validate three classes of tool compounds. These reagents will enable 1) acute, spatiotemporal inhibition of individual NaV subtypes; 2) selective fluorescent labeling of membrane NaVs; and 3) affinity purification of membrane NaVs for proteomics analysis. With the success of our research program, we will deliver a unique set of high precision chemical probes to help illuminate the complex physiology of NaVs that underlies bioelectrical signaling.

项目摘要 神经元的兴奋性依赖于严格调控的表达和离散的亚细胞 电压门控钠离子通道（NaVs）的定位。这些大的膜蛋白 复合物控制钠离子穿过细胞膜的运动， 启动和传播动作电位。希望更好地了解NaV的作用 电子信号传导中的亚型以及通道失调与 特定的人类病理（例如，心律失常、癫痫、骨骼肌疾病、神经性 pain）促使开发高精度试剂以促进NaV研究。 NaV生理学的研究目前受到缺乏可用方法的限制， 调节单个通道亚型的功能，并标记细胞内 分布、膜表达水平和结构修饰（即，蛋白质后 翻译修饰）以及对外部信号的反应。 我们正在开发用于NaV研究的小分子探针，该探针基于天然存在的双- 胍类毒素，其中石房蛤毒素（STX）是原型。这些代理的功能是 分子“软木塞”堵塞离子传导孔的细胞外口， 我们希望访问的工具化合物类型的功能。从头化学合成 使STX的修饰形式的工程，我们将使用结合蛋白质 诱变和电生理学以研究NaV对动作电位动力学的活性。 了解NaV的表达是如何被外源性因子-神经胶质细胞调节和改变的， 炎症介质，pH值，神经损伤-以及这些变化如何调节动作电位， 这是我们研究的长期目标。为了解决这些问题，我们将开发并验证 三类工具化合物。这些试剂将实现1）急性时空抑制 单个NaV亚型; 2）膜NaV的选择性荧光标记;和3）亲和力 纯化膜NaV用于蛋白质组学分析。随着我们研究的成功 计划，我们将提供一套独特的高精度化学探针，以帮助照亮 复杂的生理学基础的生物电信号。