Genetic tools to study TRPA1 channel trafficking and distribution

描述(申请人提供)：这项建议旨在产生试剂，用来可视化瞬时受体电位离子通道(TRPA1)蛋白的分布 在活着的动物身上。TRPA1在三叉神经节和背根神经节的感觉神经元中表达，在那里它是环境刺激物或导致组织损伤的化学物质的主要传感器。它也在炎症性疼痛条件下的超敏反应的发展中起着关键作用，因此被认为是疼痛疗法发展的重要靶点。然而，在进一步确定TRPA1对细胞生理学的贡献以及了解该通道在正常和炎症条件下是如何调节的一个主要障碍是，缺乏在固定组织或活组织中显示该通道蛋白的试剂。尽管有几个组织报告说 产生针对TRPA1的抗体，这些抗体通常表现出较低的特异性或敏感性。此外，抗体只能用于固定的组织或细胞培养，排除了对完整的活组织中蛋白质的研究。为了解决这一缺点，我们建议产生新的遗传探针，以可视化固定组织和活细胞中的TRPA1通道。这些探针被称为FingRs(利用mRNA显示生成的纤维连接蛋白内体)，将通过从建立在高度稳定的纤维连接蛋白骨架上的文库中选择与TRPA1结合的蛋白质来鉴定。我们预计，TRPA1 FingR将是一个极其强大的工具，可以用来检查TRPA1在健康和疾病条件下在各种细胞类型中的分布。此外，这种材料的遗传性质将使其很容易繁殖并引入活细胞。

DESCRIPTION (provided by applicant): This proposal is aimed at generating reagents with which to visualize the distribution of the transient receptor potential ion channel (TRPA1) protein in living animals. TRPA1 is expressed in sensory neurons of the trigeminal and dorsal root ganglia where it serves as the main sensor for environmental irritants or chemicals that cause tissue damage. It also plays a critical role in the development of hypersensitivity under inflammatory pain conditions, and is therefore considered an important target for the development of pain therapeutics. However, a major obstacle in further establishing the contribution of TRPA1 to cellular physiology, and in understanding how the channel is regulated under normal and inflammatory conditions, has been the lack of reagents with which to visualize the channel protein, either in fixed or in living tissue. Although several groups have reported the production of antibodies against TRPA1, these have generally exhibited low specificity or sensitivity. Moreover, antibodies can only be used in fixed tissues or in cell culture, precluding the study of the protein in intact living tissues. To address this shortcoming, we propose to generate novel genetic probes to visualize TRPA1 channels in fixed tissue and living cells. These probes, termed FingRs (fibronectin intrabodies generated with mRNA display), will be identified through a selection for proteins that bind TRPA1 from a library built on the highly stable fibronectin backbone. We anticipate that a TRPA1 FingR will be an extremely powerful tool, which could be used to examine the distribution of TRPA1 throughout the body, in a variety of cell types, in healthy and disease conditions. Moreover, the genetic nature of the material, wil allow it to be easily propagated and introduced into living cells.