Characterization of TRPM1 isoforms and binding partners in ON bipolar cells

描述(由申请人提供)：在双极细胞所在的第一视觉突触处，可靠和准确的信号对于视网膜的正常功能至关重要。视觉障碍，如先天性静止性夜盲，是光感受器到双极细胞异常信息传输的结果。最近，瞬时受体电位通道TRPM1被认为是双极细胞光反应的转导通道。TRPM1是一种非选择性阳离子通道，通过与谷氨酸受体mGluR6偶联的G蛋白信号级联，在黑暗中保持关闭。至少有10个Trpm1基因的剪接变体已经在视网膜中被鉴定出来，并且对一个主要的TRPM1亚型的纯化表明是一个二聚体。然而，TRPM1依赖的通道的组成仍然未知，剪接变体的功能作用也尚未确定。此外，通道门控G蛋白亚基的身份仍未解决。本项目的目标是验证以下假设：1)ON-双相中的功能转导通道 细胞是一个或多个TRPM1剪接变体和可能的其他蛋白质的同源或异质低聚物；2)TRPM1剪接变体与G蛋白亚基Galpha-0和Gbeta-Gamma有不同的相互作用。这些实验将用于下列特定目标：1)使用TRPM1靶向mRNA的深度测序确定新的TRPM1剪接变体并量化其在小鼠视网膜中的相对丰度；2)表征TRPM1变体在同质或异质状态下的通道功能，并利用膜片钳电生理、活细胞钙成像和免疫共沉淀等方法确定其与G蛋白亚基的相互作用；3)使用质谱仪鉴定TRPM1在视网膜中的结合伙伴，并使用超分辨显微镜和邻近连接技术确定其与体外标记的剪接变体的共同定位。这项研究将首次解决TRPM1剪接的功能作用 并研究形成功能性TRPM1依赖通道的相互作用。加深对光感受器-双极细胞突触信号转导机制的了解，将有助于确定治疗干预的新靶点，以治疗与视觉信息传输受损相关的疾病。

DESCRIPTION (provided by applicant): Reliable and precise signaling at the first visual synapse, where ON bipolar cells are located, is crucial for the proper functioning of the retina. Visual disorders such as congenital stationary night blindness are the result of aberrant information transmission from photoreceptors to bipolar cells. Recently, the transient receptor potential channel TRPM1 has been implicated as the transduction channel responsible for the light response of ON bipolar cells. TRPM1 is a nonselective cation channel, kept closed in darkness by a G-protein signaling cascade coupled to the glutamate receptor mGluR6. At least 10 splice variants of the Trpm1 gene have been identified in the retina and purification of a major TRPM1 isoform indicates a dimer. However, the composition of the TRPM1-dependent channel is still unknown and the functional role of splice variants is as yet undetermined. Furthermore, the identity of the channel gating G-protein subunit remains unresolved. The goal of this project is to test the hypotheses that 1) the functional transduction channel in ON-bipolar cells is a homo- or hetero-oligomer of one or more TRPM1 splice variants and possibly other proteins; and that 2) TRPM1 splice variants interact differentially with the G-protein subunits Galpha-0 and Gbeta-gamma. These will be tested in the following specific aims: 1) to identify new TRPM1 splice variants and quantify their relative abundance in the mouse retina, using TRPM1-targeted mRNA deep sequencing; 2) to characterize channel function of TRPM1 variants in their homomeric or heteromeric state, and determine channel interactions with G-protein subunits, using patch clamp electrophysiology, live cell Ca2+ imaging and co-immunoprecipitation and 3) to identify TRPM1 binding partners in the retina using mass spectrometry, and determine their co-localization with epitope-tagged splice variants in vitro, using super resolution microscopy and proximity ligation. This study will be the first to address the functional role of TRPM1 splice variants in the retina and investigate the interactions that form the functional TRPM1-dependent channel. Improved understanding of the signal transduction mechanisms at the photoreceptor-bipolar cell synapse will help identify novel targets for therapeutic intervention in the treatmentof diseases associated with impaired transmission of visual information.