Identifying novel thermosensitive channels via a high throughput in vivo screen

描述（由申请人提供）：感知环境温度的能力对人类的生活至关重要。哺乳动物通过在感觉神经末梢和皮肤角质形成细胞中表达的热敏受体来检测温度信号。在过去的15年里，已经确定了5个TRP通道作为主要的热受体，它们可以感知从33℃到53℃以上的各种温暖和炎热的温度。这使得我们对动物如何感知热量有了相当清晰的认识。相比之下，我们对动物如何感知低温的理解还远远不够。发现两个TRP通道（TRPM8和TRPA1）对寒冷有反应；然而，它们对冷感的贡献不大。显然，除了trp之外，冷敏感通道，特别是那些感知有害低温（<15°C）的通道，肯定存在，但尚未被发现。困难主要是由于缺乏有效的筛选系统来识别温度敏感通道。这些TRP通道作为热受体的鉴定都是通过候选基因方法实现的，但尚未发现其他TRP通道成员是冷敏感的。因此，未知的冷受体一定是由不同种类的基因编码的。与哺乳动物类似，秀丽隐杆线虫也通过热敏通道感知全方位的温度信号。有趣的是，大多数（如果不是全部的话）离子通道（如电压通道、机械通道、温度通道和配体通道）在秀丽隐杆线虫中是进化保守的。特别是那些已知的热敏通道，如TRP通道，也在秀丽隐杆线虫中发现。因此，秀丽隐杆线虫的热敏通道可能具有进化保守性。这一点，再加上其较短的生成时间（约3天）和简单而丰富的遗传工具，使秀丽隐杆线虫成为鉴定新型热敏通道的理想系统。为了克服难以捉摸的冷敏感通道克隆的困难，我们在这里开发了秀丽隐杆线虫作为一种新的高通量体内筛选系统。利用这个系统，我们已经分离出有冷感缺陷的突变体。在这个提议中，我们将首先克隆突变基因，然后在异源系统中对这些基因及其哺乳动物同源物进行表征，以测试它们是否编码冷敏感通道。这项工作可能会导致在哺乳动物中发现难以捉摸的热敏通道，这些通道检测有害的寒冷并介导冷诱发的疼痛和体温调节。此外，由于热敏通道也被其他有害线索（如有毒化学物质和/或机械刺激）激活，这些通道广泛参与疼痛感觉。因此，提出的工作也可能促进我们对疼痛感觉的整体理解，并可能有助于确定疼痛治疗的潜在治疗靶点。

DESCRIPTION (provided by applicant): The ability to sense environmental temperature is essential for human life. Mammals detect temperature cues via thermosensitive receptors expressed in sensory nerve endings and keratinocytes in the skin. Work in the past fifteen years has identified five TRP channels as the primary heat receptors that sense a full range of warm and hot temperatures, spanning from 33¿C to over 53¿C. This has led to a fairly clear understanding of how animals sense heat. By contrast, our understanding of how animals sense cold temperatures is far from complete. Two TRP channels (TRPM8 and TRPA1) are found to respond to cold; however, they only modestly contribute to cold sensation. Apparently, cold-sensitive channels other than TRPs, particularly those sensing noxious cold temperatures (<15¿C), must exist but have yet to be identified. The difficulty largely results from the lack of n efficient screening system for identifying temperature-sensitive channels. The identification of those TRP channels as thermo-receptors was all achieved through candidate gene approaches, but none of the remaining TRP channel members has been found to be cold-sensitive. Thus, the unknown cold receptors must be encoded by distinct classes of genes. Similar to mammals, C. elegans also senses a full range of temperature cues through thermosensitive channels. Interestingly, most, if not all, ion channels (e.g. voltage-, mechanically-, temperature-, and ligand-gated channels) are evolutionarily conserved in C. elegans. In particular, those known thermosensitive channels, such as TRP channels, are also found in C. elegans. Thus, thermosensitive channels in C. elegans are likely to be evolutionarily conserved. This, together with its short generation time (~3 days) and facile and rich genetic tools, makes C. elegans an ideal system for identifying novel thermosensitive channels. To circumvent the difficulty surrounding the cloning of the elusive cold- sensitive channels, here we develop C. elegans as a novel high throughput in vivo screening system. Using this system, we have isolated mutants defective in cold sensation. In this proposal, we will first clone mutant genes and then characterize these genes and their mammalian homologs in heterologous systems to test whether they encode cold-sensitive channels. The proposed work may lead to identification of the elusive thermosensitive channels that detect noxious cold and mediate cold-evoked pain and thermoregulation in mammals. Moreover, as thermosensitive channels are also activated by other noxious cues such as noxious chemicals and/or mechanical stimuli, these channels are broadly involved in pain sensation. As such, the proposed work may also facilitate our understanding of pain sensation as a whole and may help identify potential therapeutic targets for pain treatment.