Identifying novel thermosensitive channels via a high throughput in vivo screen

通过高通量体内筛选识别新型热敏通道

• 批准号: 8893182
• 负责人: Shawn Xu
• 金额: $ 31万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8893182
• 关键词: Animals; Caenorhabditis elegans; Calcium; Calcium Channel; Calcium Signaling; Candidate Disease Gene; Chemicals; Cloning; Cues; Detection; Environment; Esthesia; Fluorescence; Gene Mutation; Generations; Genes; Genetic; Genetic Screening; Genome; Health; Heating; Homologous Gene; Human; Human body; Image; Incubated; Ion Channel; Lead; Life; Mammals; Mechanics; Mediating; Mutation; Pain; Physiologic Thermoregulation; Sensory Nerve Endings; Skin; Stimulus; Syndrome; System; TRP channel; Temperature; Temperature Sense; Testing; Time; Tissues; Transgenic Organisms; Work; chronic pain; cold temperature; deep sequencing; design; genome sequencing; high throughput screening; in vivo; keratinocyte; ligand gated channel; member; mutant; nervous system disorder; novel; patch clamp; receptor; response; screening; sensor; therapeutic target; tool; voltage

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.

描述（由申请人提供）：感知环境温度的能力对于人类的生命至关重要。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热敏受体来检测温度线索。过去 15 年的工作已经确定了五个 TRP 通道作为主要的热感受器，可以感知从 33°C 到 53°C 以上的各种温暖和高温。这使得人们对动物如何感知热量有了相当清晰的了解。相比之下，我们对动物如何感知低温的理解还远未完成。两个 TRP 通道（TRPM8 和 TRPA1）被发现对寒冷有反应；然而，它们对冷感的贡献很小。显然，TRP 以外的冷敏感通道，特别是那些感测有害低温 (<15°C) 的通道，必定存在，但尚未被识别。这一困难很大程度上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，但尚未发现其余 TRP 通道成员对冷敏感。因此，未知的冷受体必须由不同类别的基因编码。与哺乳动物类似，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）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）在秀丽隐杆线虫中进化上是保守的。特别是，那些已知的热敏通道，例如 TRP 通道，也存在于秀丽隐杆线虫中。因此，线虫中的热敏通道可能在进化上是保守的。这一点，加上其较短的世代时间（约 3 天）和简便且丰富的遗传工具，使秀丽隐杆线虫成为识别新型热敏通道的理想系统。为了克服克隆难以捉摸的冷敏感通道的困难，我们开发了秀丽隐杆线虫作为一种新型的高通量体内筛选系统。利用这个系统，我们分离出了冷感缺陷的突变体。在这个提案中，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。拟议的工作可能会导致鉴定出难以捉摸的热敏通道，这些通道可以检测哺乳动物中的有害寒冷并调节寒冷引起的疼痛和体温调节。此外，由于热敏通道也会被其他有害信号（例如有毒化学物质和/或机械刺激）激活，因此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。