Activation of the ion channel TRPV1 by peptide toxins

肽毒素激活离子通道 TRPV1

基本信息

批准号：
8049048
负责人：
Christopher John Bohlen
金额：
$ 1.6万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2012-02-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8049048
关键词：
Acute Pain 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

项目摘要

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）：感官感知的第一步是将物理刺激转移到细胞信号事件中。兴奋性阳离子通道TRPV1在周围神经纤维上表达，并通过有害的热和酸性pH激活，从而充当有害外部条件的生理检测器。朱利叶斯实验室最近发现，至少一种蜘蛛剑桥诗篇，产生肽毒素，通过激活TRPV1引起疼痛和炎症。作为特定通道类型的高度选择性调节剂，已证明肽毒素是理解多个离子通道家族的结构，功能和生理学的强大工具。在此提案中，生化和电生理技术都将用于研究TRPV1毒素激活的机制。这些研究将提高我们对这一重要感觉传感器的分子理解，并为通道产生有价值的生化探针。在基础科学层面上，该项目将洞悉该重要离子通道的分子基础，并促进我们对周围神经系统中如何检测到有害刺激的分子理解。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。该项目有两个具体的目标。第一个目的是确定负责TRPV1毒素激活的位点。将从TRPV1产生嵌合体和点突变，并将测试突变通道的毒素激活。通道激活将通过钙成像和电生理学分析。同样，将生成荧光或无线电标记的香草毒素衍生物，以直接监测毒素结合。第二个目的是表征一种新型的TRPV1毒素。我发现了一种新型的TRPV1毒素激动剂，它与先前鉴定的毒素表现出极大的序列同源性。我假设，尽管序列差异有所不同，但这种新型毒素和已知的毒素已趋于趋于靶向TRPV1的同一区域。同样，新毒素具有独特的序列，与肽毒素报道的任何序列不同，该独特序列如何指示毒素功能。该项目将致力于对狼蛛毒液中的成分与辣椒素受体TRPV1相互作用的机械理解，以引起疼痛和炎症。这项工作将促进我们对有害刺激的基本感觉的分子机制的理解。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为分子水平上神经信号传导的科学理解做出重大贡献。通过这个提出的研究项目，我将研究离子渠道生理学，以深入了解分子神经科学中使用的技术和方法，并且还将熟悉生产性独立研究所需的逻辑和方法。具体而言，我将使用生化和电生理技术来研究通道毒素复合物的相互作用。我对蛋白质蛋白质相互作用的生物化学进行了一些培训，我将使用拟议的项目将此背景应用于膜环境。我还将通过特定于膜的生化技能来扩展我的技术技能。研究计划的另一部分涉及电生理技术，这对于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理论的学术理解。朱利叶斯实验室和邻近实验室成员的专业知识代表着发展这些技能的重要资产。生化和电生理技术和观点的这种结合代表了一个坚实的基础，可以追求我对神经系统中信号分子进行独立研究的目标。