Exploiting toxins to probe sensory signaling

描述(由申请人提供)：天然植物产品已成为破译与躯体感觉、伤害性感觉和疼痛有关的细胞和分子机制的极有价值的工具。值得注意的例子包括使用天然止痛剂，如吗啡(从罂粟中提取)和水杨酸盐(从柳树皮中提取)来分别发现阿片受体和环氧合酶。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着急性或慢性疼痛的药物治疗的有效或潜在靶点。植物在产生针对感觉神经元或其他可兴奋细胞的化学制剂方面并不是独一无二的。事实上，来自脊椎动物(从甲壳类到哺乳动物)的毒液代表着一个巨大的药典，它具有巨大的潜力来产生新的试剂，用来识别或表征受体、离子通道或其他有助于感觉转导的信号分子。考虑到这一点，我们最近从狼蛛中发现了一种新的多肽毒素家族，它可以激活辣椒素受体TRPV1，产生炎性疼痛(推测这是蜘蛛抵御捕食者的防御策略的一部分)。这些所谓的香草素毒素是已知的第一种与TRPV1相互作用的多肽毒素，或者就这一点而言，是兴奋性Trp通道家族的任何成员。这一建议建立在我们对香草素的初步发现的基础上，目的是利用天然毒素和合成衍生物来探索初级传入神经元或其他类型的可兴奋细胞表达的色氨酸通道或其他受体的结构和/或功能。这项建议的第一个目标是利用香草毒素作为新的生化工具来探索TRPV1的通道结构，首先要设计实验来绘制香草毒素-TRPV1相互作用的位置图。目前对Trp通道结构或门控机制知之甚少，将我们的发现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。第二和第三个目标是针对TRPV1或可兴奋细胞上的其他部位的各种有毒生物的新型毒素进行识别和表征。这将扩大用于研究已知或新的感觉受体的药理学试剂的范围，包括那些对非常重要但仍然神秘的机械转导过程做出贡献的药物。细胞表面受体和离子通道在全身细胞通讯中发挥着重要作用，包括神经系统和心血管功能的许多方面。在这项建议中，我们描述了利用天然毒素作为新的生化工具来研究受体和离子通道的结构和功能的策略，这些受体和离子通道有助于检测感觉刺激，特别是与疼痛感觉有关的刺激。这项工作的结果将提供一个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伤害性(产生疼痛)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治疗慢性疼痛综合征的新治疗剂或方案的策略提供洞察力。

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.