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的可靠和精确的信号传导对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TRPM1被认为是负责ON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。在视网膜中发现了至少10个Trpm1基因的剪接变体，纯化的主要Trpm1异构体表明其为二聚体。然而，trpm1依赖性通道的组成仍然未知，剪接变异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确定。本项目的目的是验证以下假设：1)ON-bipolar的功能转导通道

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.