Activation of the ion channel TRPV1 by peptide toxins

描述(申请人提供)：感官知觉的第一步是将物理刺激转化为细胞信号事件。兴奋性阳离子通道TRPV1在外周神经纤维上表达，并被伤害性高温和酸性pH激活，从而作为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，Psalmopoeus cambridge gei，会产生多肽毒素，通过激活TRPV1引起疼痛和炎症。作为特定通道类型的高选择性调节剂，多肽毒素已被证明是了解几个离子通道家族的结构、功能和生理的强大工具。在这项建议中，将使用生化和电生理技术来研究TRPV1毒素激活的机制。这些研究将促进我们对这一重要的感觉转导的分子理解，并为该通道产生有价值的生化探针。在基础科学层面，这个项目将深入了解这一重要离子通道的分子基础，并促进我们对周围神经系统如何检测有害刺激的分子理解。从长远来看，这些努力将有助于了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进行检测。此外，还将产生香草毒素的荧光或放射性标记的衍生物，以直接监测毒素的结合。第二个目的是鉴定一种新的TRPV1毒素。我发现了一种新的毒素激动剂TRPV1，它与之前发现的毒素序列几乎没有同源性。我推测，尽管在序列上存在差异，但这种新毒素和已知的毒素已经收敛地进化到针对TRPV1的同一区域。此外，这种新的毒素有一个独特的序列，不同于已报道的任何肽毒素序列，这个独特的序列如何决定毒素的功能将被研究。这个项目将致力于从机制上理解狼蛛毒液中的成分如何与辣椒素受体TRPV1相互作用，从而导致疼痛和炎症。这项工作将促进我们对有害刺激感觉不足的分子机制的理解。从长远来看，这项研究将有助于了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。在这个拟议的研究项目中，我将学习离子通道生理学，以深入了解分子神经科学中使用的技术和方法，并熟悉富有成效的独立研究所必需的逻辑和方法。具体地说，我将使用生化和电生理学技术来研究经络毒素复合体的相互作用。我在蛋白质-蛋白质相互作用的生物化学方面接受过一些培训，我将利用拟议的项目将这一背景应用到膜环境中。我还将扩展我的技术技能，既有膜特有的技能，也有一般的生化技能。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。我将利用各种录音配置来整合我对电生理学理论的学术理解。朱利叶斯实验室和邻近实验室成员的专业知识是开发这些技能的巨大财富。这种生化和电生理技术和观点的结合为我实现在神经系统中进行信号分子独立研究的目标奠定了坚实的基础。

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.