Characterization of TRPM1 isoforms and binding partners in ON bipolar cells

描述（由申请人提供）：ON双极细胞所在的第一视觉突触处的可靠且精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症，是光感受器向双极细胞传递异常信息的结果。最近，瞬时受体电位通道TRPM 1已被认为是负责ON双极细胞光反应的转导通道。TRPM 1是一种非选择性阳离子通道，在黑暗中通过与谷氨酸受体mGluR 6偶联的G蛋白信号级联保持关闭。至少有10个剪接变体的Trpm 1基因已被确定在视网膜和纯化的主要TRPM 1亚型表明二聚体。然而，TRPM 1依赖性通道的组成仍然是未知的，剪接变体的功能作用尚未确定。此外，通道门控G蛋白亚基的身份仍然没有得到解决。本研究的目的是验证以下假说：1）ON-双极细胞中的功能性转导通道 细胞是一个或多个TRPM 1剪接变体和可能的其他蛋白质的同源或异源寡聚体; 2）TRPM 1剪接变体与G蛋白亚基G α-0和G β-γ有差异地相互作用。这些将在以下具体目标中进行测试：1）使用TRPM 1靶向mRNA深度测序来鉴定新的TRPM 1剪接变体并量化它们在小鼠视网膜中的相对丰度; 2）使用膜片钳电生理学表征TRPM 1变体在其同聚或异聚状态下的通道功能，并确定通道与G蛋白亚基的相互作用，活细胞Ca 2+成像和免疫共沉淀，以及3）使用质谱法鉴定视网膜中的TRPM 1结合配偶体，并使用超分辨率显微镜和邻近连接法在体外确定它们与表位标记的剪接变体的共定位。这项研究将是第一个解决TRPM 1剪接的功能作用， 在视网膜中的变体，并调查形成功能TRPM 1依赖通道的相互作用。对光感受器-双极细胞突触的信号传导机制的进一步了解将有助于确定治疗与视觉信息传递受损相关的疾病的治疗干预的新靶点。

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.