Exploiting toxins to probe sensory signaling

描述（由申请人提供）：天然植物产品已成为解释躯体感觉、伤害感受和疼痛的细胞和分子机制的非常有价值的工具。值得注意的例子包括使用天然镇痛剂，如吗啡（来自罂粟）和水杨酸盐（来自杨柳皮），分别发现阿片受体和环氧化酶。其他重要的例子包括使用天然刺激物，如辣椒素（来自辣椒）和薄荷醇（来自薄荷叶），以识别分别检测热和冷的离子通道。事实上，这些蛋白质中的每一种都代表了急性或慢性疼痛的药理学管理的经验证的或潜在的靶标。植物并不是唯一能够产生针对感觉神经元或其他可兴奋细胞的化学物质的植物。事实上，来自脊椎动物生物体（从甲壳类动物到哺乳动物）的毒液代表了巨大的药典，其具有产生新试剂的巨大潜力，所述新试剂用于鉴定或表征有助于感觉转导的受体、离子通道或其他信号分子。考虑到这一点，我们最近从狼蛛中发现了一个新的肽毒素家族，它能激活辣椒素受体TRPV 1，产生炎性疼痛（可能是蜘蛛抵御捕食者的防御策略的一部分）。这些所谓的香草毒素是已知的第一种与TRPV 1相互作用的肽毒素，或者就此而言，与兴奋性TRP通道扩展家族的任何成员相互作用。该建议建立在我们最初发现的香草毒素的基础上，目的是使用天然毒素和合成衍生物来探测初级传入神经元或其他类型的可兴奋细胞表达的TRP通道或其他受体的结构和/或功能。该建议的第一个目的是面向开发的香草毒素作为新的生化工具，以探测TRPV 1通道结构，开始设计的实验，以映射香草毒素-TRPV 1相互作用的网站。目前对TRP通道结构或门控机制知之甚少，将我们的研究结果与电压门控通道领域的数据进行比较，将为这些和相关的结构-功能问题提供有价值的见解。第二个和第三个目标是针对识别和表征来自各种有毒生物的新型毒素，这些毒素靶向TRPV 1或可兴奋细胞上的其他位点。这将扩大药理学试剂的库，用于研究已知或新的感觉受体，包括那些有助于非常重要但仍然神秘的机械转导过程的受体。细胞表面受体和离子通道在整个身体的细胞通讯中起着至关重要的作用，包括神经系统和心血管功能的许多方面。在这个建议中，我们描述了利用天然毒素作为新的生化工具，研究受体和离子通道的结构和功能，有助于检测感官刺激，特别是与疼痛感觉的关系的战略。这项工作的结果将提供更深入的了解这些受体和通道如何在正常和病理条件下对有害（疼痛产生）刺激做出反应，所有这些都与阐明导致急性和慢性疼痛的过程有关。这些研究的结果可能为开发新的治疗药物或治疗慢性疼痛综合征的方案提供见解。

DESCRIPTION (provided by applicant): Natural plant products have served as tremendously valuable tools for deciphering cellular and molecular mechanisms contributing to somatosensation, nociception, and pain. Notable examples include the use of natural analgesics, such as morphine (from the opium poppy) and salicylate (from willow bark) to discover opioid receptors and cyclooxgenases, respectively. Other important examples include the use of natural irritants, such as capsaicin (from chili peppers) and menthol (from mint leaves) to identify ion channels that detect heat and cold, respectively. Indeed, each of these proteins represents a validated or potential target for pharmacological management of acute or chronic pain. Plants are not unique in their capacity to produce chemical agents that target sensory neurons or other excitable cells. Indeed, venoms from vertebrate organisms (ranging from crustaceans to mammals) represent a vast pharmacopoeia that has great potential to yield novel agents with which to identify or characterize receptors, ion channels, or other signaling molecules that contribute to sensory transduction. With this in mind, we have recently discovered a novel family of peptide toxins from tarantula that activate the capsaicin receptor, TRPV1, to produce inflammatory pain (presumably as part of the spider's defensive strategy to ward off predators). These so-called vanillotoxins are the first peptide toxins known to interact with TRPV1, or for that matter, any member of the extended family of excitatory TRP channels. This proposal builds on our initial discovery of the vanillotoxins, with the goal of using natural toxins and synthetic derivatives to probe the structure and/or function of TRP channels or other receptors expressed by primary afferent neurons or other types of excitable cells. The first aim of this proposal is geared towards exploiting the vanillotoxins as novel biochemical tools with which to probe TRPV1 channel structure, beginning with experiments designed to map sites of vanillotoxin-TRPV1 interaction. Little is currently known about TRP channel architecture or gating mechanisms, and comparison of our findings with data from the voltage-gated channel field will provide valuable insights into these and related structure-function questions. The second and third aims are directed towards identifying and characterizing novel toxins from a variety of venomous creatures that target TRPV1 or other sites on excitable cells. This will expand the repertoire of pharmacological agents with which to study known or novel sensory receptors, including those that contribute to the very important, but still enigmatic process of mechanotransduction. PUBLIC HEALTH RELEVANCE Cell surface receptors and ion channels play essential roles in cellular communication throughout the body, including many aspects of nervous system and cardiovascular function. In this proposal, we describe strategies for exploiting natural toxins as new biochemical tools with which to study the structure and function of receptors and ion channels that contribute to the detection of sensory stimuli, particularly in relationship to pain sensation. Results from this work will provide a more in-depth understanding of how these receptors and channels respond to noxious (pain-producing) stimuli under normal and pathological conditions, all of which is relevant to elucidating processes that contribute to acute and chronic pain. Results from these studies may provide insights into strategies for developing novel therapeutic agents or protocols for the treatment of chronic pain syndromes.