Identifying novel thermosensitive channels via a high throughput in vivo screen

描述(申请人提供)：感知环境温度的能力对人类生活是必不可少的。哺乳动物通过感觉神经末梢和皮肤角质形成细胞中表达的温度敏感受体来检测温度信号。过去15年的工作已经确定了5个Trp通道作为主要的热感受器，它们可以感知从33�C到53�C的全方位的温暖和高温。这使得我们对动物如何感知热量有了相当清晰的理解。相比之下，我们对动物如何感知寒冷温度的理解还远未完成。有两个Trp通道(TRPM8和TRPA1)对冷有反应，但它们对冷感的贡献不大。显然，除TRPS以外的其他冷敏感通道，特别是那些感知有害低温(&lt；15�C)的通道，肯定存在，但尚未被识别。困难的主要原因是缺乏有效的筛选系统来识别温度敏感通道。这些Trp通道都是通过候选基因方法被鉴定为热受体，但其余的Trp通道成员都没有被发现是冷敏感的。因此，未知的寒冷受体必须由不同类别的基因编码。与哺乳动物类似，线虫也通过温度敏感通道感知各种温度信号。有趣的是，大多数离子通道(例如电压门控、机械门控、温度门控和配基门控通道)在线虫中进化上是保守的。特别是，那些已知的温度敏感通道，如Trp通道，也在线虫中发现。因此，线虫的温度敏感通道在进化上可能是保守的。这一点，再加上其短的世代时间(~3天)和方便丰富的遗传工具，使线虫成为识别新的温度敏感通道的理想系统。为了绕过克隆难以捉摸的冷敏感通道的困难，我们在这里开发了线虫作为一种新的高通量体内筛选系统。利用这个系统，我们已经分离出了有冷感缺陷的突变体。在这个方案中，我们将首先克隆突变基因，然后在异源系统中表征这些基因及其哺乳动物同源物，以测试它们是否编码冷敏感通道。这项拟议的工作可能会导致识别难以捉摸的温度敏感通道，这些通道可以检测有害的寒冷，并在哺乳动物中介导冷诱发的疼痛和体温调节。此外，由于温度敏感通道也被其他有害信号激活，如有毒化学物质和/或机械刺激，这些通道广泛参与痛觉。因此，拟议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.