Activation of the ion channel TRPV1 by peptide toxins

描述（由申请人提供）：感官知觉的第一步是将物理刺激转导成细胞信号事件。兴奋性阳离子通道TRPV1在周围神经纤维上表达，在有害热量和酸性pH下被激活，从而作为外部有害条件的生理探测器。朱利叶斯实验室最近发现，至少有一种蜘蛛，cambridgei，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、功能和生理的有力工具。本研究将采用生物化学和电生理技术研究TRPV1毒素激活的机制。这些研究将促进我们对这种重要的感觉传感器的分子理解，并为通道产生有价值的生化探针。在基础科学水平上，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是如何被检测到的分子理解。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TRPV1毒素激活的位点。将从TRPV1产生嵌合体和点突变，并对突变通道进行毒素激活测试。通道激活将通过钙成像和电生理来检测。此外，将产生香草毒素的荧光或放射性标记衍生物，以直接监测毒素结合。第二个目标是表征一种新的TRPV1毒素。我发现了一种新型的TRPV1毒素激动剂，它与之前发现的毒素几乎没有序列同源性。我推测，尽管在序列上存在差异，但这种新毒素和已知毒素已经进化趋同，以靶向TRPV1的同一区域。此外，新毒素具有独特的序列，不像任何已报道的肽毒素序列，以及这种独特的序列如何决定毒素的功能将被研究。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TRPV1相互作用，从而引起疼痛和炎症。这项工作将促进我们对有害刺激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慢性疼痛综合征。

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.