Exploiting toxins to probe sensory signaling

描述（由申请人提供）：天然植物产品已经成为非常有价值的工具，用于破译身体感觉、伤害感觉和疼痛的细胞和分子机制。值得注意的例子包括使用天然镇痛药，如吗啡（来自罂粟）和水杨酸（来自柳树皮），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）来识别分别探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有效或潜在目标。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，来自动物（从蛛形纲动物到哺乳动物）的毒液代表了一个巨大的药典，它具有巨大的潜力，可以产生用于识别或表征受体、离子通道或其他有助于感觉转导的信号分子的新药。事实上，在之前的资助期内，我们确定了两种这样的毒素——一种来自蜘蛛，另一种来自蛇——它们分别作为TRPV1和ASIC1通道的新型、强效和高选择性激动剂。在每种情况下，这些毒素都能在原子分辨率下阐明通道的激活、完全开放状态，为通道门控和调制的结构机制提供前所未有的见解。该提案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型体感受体的药理学试剂的曲目。第一个目的是集中描述我们在基于感觉神经元的筛选试验中发现的两种蜘蛛毒素，它们针对由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通道域(s)指定毒素敏感性，并说明其

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.