Guanidinium Toxins as Molecular Probes for NaV Study

胍毒素作为 NaV 研究的分子探针

基本信息

批准号：
10618785
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
项目类别：
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/10618785
关键词：
Action Potentials Acute Address Affect Affinity Affinity Chromatography Antibodies Arrhythmia Binding Binding Proteins Biological Phenomena Biotin Biotinylation 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 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 Natural Source 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 Structure Study models Surface Tetrodotoxin Therapeutic Tissues Toxin Training Transfection Vestibule Work bioelectricity cell fixing cell type chemical synthesis experimental study extracellular genetic approach 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

项目摘要

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.

项目摘要神经元的刺激性取决于严格调节的表达和离散的亚细胞电压门控钠离子通道（NAVS）的定位。这些大膜蛋白复合物控制钠离子跨细胞膜的运动，并负责发起和传播行动潜力。渴望更好地了解NAV的作用电信号传导中的亚型以及通道失调和特定的人类病理（例如心律不齐，癫痫，骨骼肌疾病，神经性疾病疼痛）激发高精度试剂的发展以促进NAV研究。 NAV生理学的调查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的变化分布，膜表达水平和结构修饰（即蛋白在活细胞和外部线索中的转化修饰）。我们正在为基于天然发生的双h- 鸟苷毒素，其中萨克西毒素（STX）是原型。这些代理作用分子“软木塞”，以阻塞离子电导孔的细胞外口，是理想的我们希望访问的工具化合物类型的功能。从头化学合成启用了修改形式的STX的工程，我们将与蛋白质结合使用诱变和电生理学研究了对动作电位动力学的NAV活性。了解如何通过外在因素（乳细胞）调节和改变NAV表达炎症介质，pH，神经损伤以及这种变化如何调节动作电位是我们研究的长期目标。为了解决这些问题，我们将开发和验证三类工具化合物。这些试剂将启用1）急性时空抑制单个导航子类型； 2）膜导航的选择性荧光标记； 3）亲和力用于蛋白质组学分析的膜NAV的纯化。随着我们的研究成功程序，我们将提供一套独特的高精度化学问题，以帮助照亮基于生物电信信号传导的NAV的复杂生理。