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生活至关重要。哺乳动物通过在皮肤中的感觉神经末梢和角质形成细胞中表达的热敏受体来检测温度线索。在过去的十五年中，研究人员已经确定了五个TRP通道作为主要的热受体，它们可以感受到从33 ℃到53 ℃以上的各种温暖和高温。这使得我们对动物如何感知热量有了相当清楚的了解。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。两个TRP通道（TRPM 8和TRPA 1）被发现对冷有反应;然而，它们仅适度地促进冷感觉。显然，除了TRP之外的冷敏感通道，特别是那些感知有害低温（<15 ° C）的通道，必须存在，但尚未确定。这一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通道作为热感受器的鉴定都是通过候选基因方法实现的，但是没有发现剩余的TRP通道成员是冷敏感的。因此，未知的冷受体必须由不同类别的基因编码。与哺乳动物相似，C.秀丽线虫还通过热敏通道感知全方位的温度线索。有趣的是，大多数（如果不是全部的话）离子通道（例如电压门控通道、机械门控通道、温度门控通道和配体门控通道）在C中是进化保守的。优雅特别是，那些已知的热敏通道，如TRP通道，也在C.优雅因此，C.在进化上是保守的。这一点，再加上它的短世代时间（~3天）和方便和丰富的遗传工具，使C。elegans是鉴定新型热敏通道的理想系统。为了克服克隆这种难以捉摸的冷敏感通道的困难，我们在这里开发了C. elegans作为一种新的高通量体内筛选系统。利用这个系统，我们分离出了有冷感觉缺陷的突变体。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。这项工作可能会导致识别难以捉摸的热敏通道，检测有害的寒冷和介导冷诱发的疼痛和温度调节哺乳动物。此外，由于热敏通道也被其他有害线索激活，如有害化学物质和/或机械刺激，这些通道广泛参与疼痛感觉。因此，拟议的工作也可能有助于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.