TRP-CHANNELS IN LENS DEVELOPMENT AND CATARACT

项目总结/摘要 非选择性阳离子通道的瞬时受体电位（TRP）超家族在多种生物学行为中起着关键作用。 生理过程，包括对光、温度、压力和疼痛的感知。在过去的遗传 研究发现，melastatin相关的TRP通道（TRPM 3）成员的突变 是一种遗传性小儿白内障的基础，与青光眼和眼前节相关 缺陷在本研究中，我们将研究TRPM 3在透镜发育和白内障病理学中的作用 使用基因编辑的小鼠和细胞系，解决三个具体目标。在aim-1中，我们将表征透镜 通过RNA深度测序的TRP通道转录本剪接变体和钙通道特性 透镜TRPM 3亚型的荧光钙成像技术。在aim-2中，我们将描述 TRPM 3功能障碍和缺乏对透镜阳离子状态、水含量和钙内流性质的影响 使用原子吸收光谱法和钙成像技术的组合。在aim-3中，我们将 表征TRPM 3功能障碍和缺乏对透镜形态发生和基因表达的影响 分别使用差异免疫荧光显微镜和RNA深度测序技术。 这些研究的结果将提供关于TRP通道在透镜生长、阳离子交换和细胞增殖中的作用的新见解。 儿童白内障背景下的体内平衡、钙动力学和病理生理学--一项临床研究 终身视力损害的重要危险因素。更广泛地说，这些研究将有助于 了解其他眼组织中的TRP通道功能和功能障碍（例如，虹膜、睫状体）和 疾病（例如，青光眼、前眼缺损）。

Project Summary/Abstract The transient receptor potential (TRP) superfamily of non-selective cation channels play critical roles in diverse physiological processes including the perception of light, temperature, pressure, and pain. In previous genetic studies, we have discovered that mutation of a member of the melastatin-related TRP-channels (TRPM3) underlies an inherited form of pediatric cataract that is variably associated with glaucoma and anterior segment defects. In this proposal, we will investigate the role of TRPM3 in lens development and cataract pathobiology using gene-edited mice and cell-lines by addressing three specific aims. In aim-1, we will characterize lens TRP-channel transcript splice-variants by means of RNA deep-sequencing and the calcium channel properties of lens TRPM3 isoforms using fluorometric calcium imaging techniques. In aim-2, we will characterize the effects of TRPM3 dysfunction and deficiency on lens cation status, water content, and calcium influx properties using a combination of atomic absorption spectrometry and calcium imaging techniques. In aim-3, we will characterize the effects of TRPM3 dysfunction and deficiency on lens morphogenesis and gene expression using differential immuno-fluorescence microscopy and RNA deep-sequencing techniques, respectively. Results from these studies will provide new insights regarding the role of TRP-channels in lens growth, cation homeostasis, calcium dynamics, and pathophysiology within the context of pediatric cataract – a clinically important risk factor for lifelong visual impairment. More broadly, these studies will contribute to an understanding of TRP-channel function and dysfunction in other eye tissues (e.g., iris, ciliary body) and diseases (e.g., glaucoma, anterior eye defects), respectively.