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，来自动物(从蜘蛛纲动物到哺乳动物)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。事实上，在之前的资助期间，我们发现了两种这样的毒素--一种来自蜘蛛，另一种来自蛇--分别作为TRPV1和ASIC1通道的新颖、有效和高选择性的激动剂。在每种情况下，这些毒素都能够在原子分辨率下阐明激活的、完全开放的通道状态，为通道门控和调制的结构机制提供了前所未有的见解。这项建议建立在我们在毒素发现和表征方面的成功和专业知识的基础上，目标是扩大用于研究已知或新的体感受体的药理药剂的范围。第一个目标是鉴定两种蜘蛛毒素，这两种毒素是我们在基于感觉神经元的筛选实验中鉴定的，它们针对这些细胞表达的特定电压门控钠通道(NAV)亚型。我们建议确定NAV通道结构域(S)，它指定毒素敏感性并解释其 亚型选择性。此外，我们将使用小鼠遗传学在体内测试毒素的选择性，以及 在细胞和行为模式中确定介导这些毒素的兴奋和致过敏作用的感觉神经元亚群。第二个目标是鉴定两种新的毒素--一种来自蜈蚣，另一种来自蛇--我们也通过功能筛选鉴定了这两种毒素，它们激活了初级传入感觉神经元，在小鼠身上引发了伤害性反应。我们建议确定这些毒素的分子靶点，并确定它们激活疼痛通路感觉神经元的信号机制。这些研究将发现新的感觉转导分子和/或提供强大的新工具来确定已知的转导如何调节伤害性感受器的兴奋性。从这项工作中收集的信息将为止痛药的开发提供重要的药理学线索和见解。

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 animals (ranging from arachnids 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. Indeed, in the previous funding period we identified two such toxins - one from spider and another from snake - that serve as novel, potent, and highly selective agonists for TRPV1 and ASIC1 channels, respectively. In each case, these toxins enabled elucidation of the activated, fully open state of the channel at atomic resolution, providing unprecedented insights into structural mechanisms underlying channel gating and modulation. This proposal builds on our success and expertise in toxin discovery and characterization, with the goal of expanding the repertoire of pharmacological agents with which to study known or novel somatosensory receptors. The first aim is focused on characterizing two spider toxins that we identified in a sensory neuron- based screening assay, and which target a specific voltage-gated sodium channel (Nav) subtype expressed by these cells. We propose to identify the Nav channel domain(s) that specifies toxin sensitivity and accounts for its subtype selectivity. Furthermore, we shall test toxin selectivity in vivo using mouse genetics, and identify the subpopulation of sensory neurons that mediate the excitatory and algogenic actions of these toxins in cellular and behavioral paradigms. The second aim is focused on characterizing two novel toxins - one from centipede and the other from snake - that we also identified by functional screening, and which activate primary afferent sensory neurons to elicit nocifensive responses in mice. We propose to identify the molecular targets of these toxins and determine the signaling mechanisms through which they activate sensory neurons of the pain pathway. These studies will uncover novel sensory transduction molecules and/or provide powerful new tools for determining how known transducers work to modulate nociceptor excitability. Information gleaned from this work will provide important pharmacologic leads and insights pertinent to the development of analgesic agents.