Activation of the ion channel TRPV1 by peptide toxins

肽毒素激活离子通道 TRPV1

• 批准号: 7896800
• 负责人: Christopher John Bohlen
• 金额: $ 3.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7896800
• 关键词: Acute; Affinity; Agonist; Amino Acid Sequence; Basic Science; Binding; Biochemical; Biochemistry; Biological Assay; Burn injury; Calcium; Capsaicin; Cations; Characteristics; Chemicals; Chili Pepper; Chimera organism; Complex; Cysteine; Electrophysiology (science); Environment; Esthesia; Event; Exhibits; Family; Foundations; Future; Goals; Heating; Image; Individual; Inflammation; Ion Channel; Irritants; Label; Location; Logic; Longitudinal Studies; Membrane; Molecular; Monitor; Nerve Fibers; Nervous system structure; Neurosciences; Pain; Peptides; Perception; Peripheral Nerves; Peripheral Nervous System; Physiological; Physiology; Point Mutation; Radio; Recombinants; Reporting; Research; Research Project Grants; Sensory; Sequence Homology; Signal Transduction; Signaling Molecule; Site; Solid; Spiders; Stimulus; Structure; Syndrome; TRPV1 gene; Tarantula Venoms; Targeted Toxins; Techniques; Testing; Toxin; Training; Transducers; Vanilloid; Venoms; Voltage-Gated Potassium Channel; Work; capsaicin receptor; career; chronic pain; detector; inhibitor/antagonist; insight; member; mutant; novel; prevent; protein protein interaction; relating to nervous system; scaffold; sensor; sensory system; skills; theories; tool; voltage

DESCRIPTION (provided by applicant): The first step of sensory perception is the transduction of physical stimuli into cellular signaling events. The excitatory cation channel TRPV1 is expressed on peripheral nerve fibers and is activated by noxious heat and acidic pH, thereby serving as a physiological detector of harmful external conditions. The Julius lab recently discovered that at least one species of spider, Psalmopoeus cambridgei, produces peptide toxins that elicit pain and inflammation through activation of TRPV1. As highly selective modulators of particular channel-types, peptide toxins have proven to be powerful tools for understanding the structure, function, and physiology of several ion channel families. In this proposal both biochemical and electrophysiological techniques will be used to investigate the mechanism of toxin activation of TRPV1. These studies will advance our molecular understanding of this important sensory transducer and generate valuable biochemical probes for the channel. At the basic science level, this project will bring insight into the molecular underpinnings of this important ion channel and advance our molecular understanding of how noxious stimuli are detected in the peripheral nervous system. These efforts will contribute, in the long term, towards understanding and controlling acute and chronic pain syndromes. The project has two specific aims. The first aim is to determine the sites responsible for toxin activation of TRPV1. Chimeras and point mutations will be generated from TRPV1 and mutant channels will be tested for toxin activation. Channel activation will be assayed by both calcium imaging and electrophysiology. Also, fluorescent- or radio-labeled derivatives of the vanillotoxins will be generated to directly monitor toxin binding. The second aim is to characterize a novel TRPV1 toxin. I have discovered a novel toxin agonist of TRPV1 that exhibits strikingly little sequence homology with the previously identified toxins. I hypothesize that despite the differences in sequence, this novel toxin and the known toxins have evolved convergently to target the same region of TRPV1. Also, the new toxin has a unique sequence, unlike any sequence that has been reported for a peptide toxin, and how this unique sequence dictates toxin function will be investigated. This project will work toward a mechanistic understanding of how components in tarantula venom interact with the capsaicin receptor, TRPV1, in order to cause pain and inflammation. This work will advance our understanding of the molecular mechanisms that underly sensation of noxious stimuli. In the long-term, this study will contribute towards understanding and controlling acute and chronic pain syndromes. My long-term career goal is to make significant contributions to the scientific understanding of neural signaling at the molecular level. With this proposed research project, I will study ion channel physiology to gain an intimate understanding of the techniques and approaches used in molecular neuroscience, and I will also become familiar with the logic and approaches necessary for productive independent research. Specifically, I will use both biochemical and electrophysiological techniques to study the interactions of a channel-toxin complex. I have some training in the biochemistry of protein-protein interactions, and I will use the proposed project to apply this background to the membrane environment. I will also expand my technical skillset, with both membrane-specific and general biochemical skills. Another portion of the research plan involves electrophysiological techniques, which are exceptionally important for studying neural signaling. I will utilize a variety of recording configurations to integrate my scholastic understanding of electrophysiological theory. The expertise of members of the Julius lab and neighboring labs represents a great asset in developing these skills. This combination of biochemical and electrophysiological techniques and perspectives represents a solid foundation from which to pursue my goals of conducting independent research on signaling molecules in the nervous system.

描述（由申请人提供）：感官知觉的第一步是将物理刺激转导为细胞信号事件。兴奋性阳离子通道TRPV 1在外周神经纤维上表达，并被有害热和酸性pH激活，从而充当有害外部条件的生理检测器。Julius实验室最近发现，至少有一种蜘蛛，即剑桥诗篇（Psalmopoeus cambridgei），会产生肽毒素，通过激活TRPV 1引起疼痛和炎症。作为特定离子通道类型的高选择性调节剂，肽毒素已被证明是了解几种离子通道家族的结构、功能和生理学的有力工具。本研究拟采用生物化学和电生理技术研究TRPV 1的毒素激活机制。这些研究将推进我们对这一重要感觉传感器的分子理解，并为该通道产生有价值的生化探针。在基础科学层面，该项目将深入了解这一重要离子通道的分子基础，并推进我们对周围神经系统中有害刺激如何检测的分子理解。从长远来看，这些努力将有助于理解和控制急性和慢性疼痛综合征。该项目有两个具体目标。第一个目标是确定负责TRPV 1毒素激活的位点。嵌合体和点突变将从TRPV 1产生，突变通道将用于毒素激活测试。将通过钙成像和电生理学测定通道激活。此外，将产生香草毒素的荧光或放射性标记的衍生物以直接监测毒素结合。第二个目的是表征一种新的TRPV 1毒素。我发现了一种新的TRPV 1毒素激动剂，它与以前鉴定的毒素具有惊人的小序列同源性。我推测，尽管在序列上存在差异，这种新的毒素和已知的毒素已经趋同地进化到靶向TRPV 1的相同区域。此外，新的毒素具有独特的序列，不同于已报道的肽毒素的任何序列，并且将研究这种独特的序列如何决定毒素功能。该项目将致力于了解狼蛛毒液中的成分如何与辣椒素受体TRPV 1相互作用，以引起疼痛和炎症。这项工作将促进我们对伤害性刺激感受的分子机制的理解。从长远来看，这项研究将有助于理解和控制急性和慢性疼痛综合征。 我的长期职业目标是在分子水平上对神经信号的科学理解做出重大贡献。通过这个拟议的研究项目，我将研究离子通道生理学，以深入了解分子神经科学中使用的技术和方法，我也将熟悉生产性独立研究所需的逻辑和方法。具体来说，我将使用生物化学和电生理技术来研究通道毒素复合物的相互作用。我在蛋白质-蛋白质相互作用的生物化学方面受过一些训练，我将利用所提出的项目将这种背景应用于膜环境。我还将扩大我的技术技能，包括膜特异性和一般生物化学技能。研究计划的另一部分涉及电生理技术，这对研究神经信号非常重要。我将利用各种记录配置来整合我对电生理学理论的学术理解。Julius实验室和邻近实验室成员的专业知识是发展这些技能的重要资产。这种生物化学和电生理技术和观点的结合为我实现对神经系统中信号分子进行独立研究的目标奠定了坚实的基础。