Genetic tools to study TRPA1 channel trafficking and distribution

描述（由申请人提供）：本提案旨在生成用于可视化瞬时受体电位离子通道（TRPA 1）蛋白分布的试剂 活的动物。TRPA1在三叉神经和背根神经节的感觉神经元中表达，在那里它作为引起组织损伤的环境刺激物或化学物质的主要传感器。它还在炎性疼痛条件下的超敏反应的发展中起关键作用，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TRPA 1对细胞生理学的贡献，以及在理解通道在正常和炎症条件下如何调节方面的主要障碍是缺乏试剂来可视化固定或活组织中的通道蛋白。虽然有几个团体报告说， 尽管这些方法用于生产针对TRPA 1的抗体，但它们通常表现出低特异性或灵敏度。此外，抗体只能用于固定的组织或细胞培养，排除了在完整的活组织中研究蛋白质。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。这些探针称为FingRs（用mRNA展示产生的纤连蛋白胞内抗体），将通过从建立在高度稳定的纤连蛋白骨架上的文库中选择结合TRPA 1的蛋白质来鉴定。我们预计，TRPA1 FingR将是一个非常强大的工具，可用于检查TRPA1在整个身体中的分布，在各种细胞类型中，在健康和疾病条件下。此外，材料的遗传性质将允许其容易地繁殖并引入活细胞中。

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.