TRP channel expression and function in ON-bipolar cells

描述（由申请人提供）：先天性静止性夜盲（CSNB）是一组以暗视觉受损为特征的非进行性视网膜疾病。许多基因的突变已被证明与CSNB有关。它们通常影响光感受器和二级双极细胞之间的突触传递。当突变基因仅在视杆细胞或视杆双极细胞中表达时，表型仅限于夜盲症。然而，当视锥细胞和视锥双极细胞之间的突触传递也需要突变基因功能时，进一步的视觉症状是明显的，如近视、远视、眼球震颤和视力下降。一个这样的例子是GRM 6（编码mGluR 6的基因）中的突变，其导致CSNB的常染色体隐性形式。 在视网膜中，视觉信息被分离成响应于光强度的增加和减少的通路。在第一视网膜突触处，从感光体末梢紧张性释放谷氨酸在黑暗中维持高突触浓度，其响应于光而降低。两种类型的突触后细胞，ON双极细胞和OFF双极细胞，对光感受器释放的谷氨酸产生相反的极性反应，从而建立相反的视觉通路。OFF-双极细胞中的信号传导的基础依赖于离子型谷氨酸受体的激活，这是很好理解的。然而，在ON-双极细胞中产生光反应的信号通路更为复杂，其分子机制仍有待阐明。ON-双极细胞信号传导途径起源于独特的代谢型谷氨酸受体mGluR 6，其在ON-双极细胞的树突上发现。mGluR 6通过G蛋白Go调节未识别阳离子通道的活性，使得突触谷氨酸的光诱导减少打开通道并使细胞去极化。 最近，据报道，阿帕卢萨马的CSNB与导致TRPM 1阳离子通道表达减少的突变相关。我们假设TRPM 1和其他可能相关的TRP通道是与mGluR 6偶联的阳离子通道，介导ON双极细胞的去极化光反应。我们进一步认为TRP通道的突变会导致CSNB。使用生物化学，免疫组织化学和电生理学方法的组合，我们将通过回答以下问题来测试这一假设：1。哪些TRP通道变体在ON双极细胞中表达？2.在双极细胞中表达的TRP通道中携带无效突变的小鼠是否患有CSNB？3.视网膜ON双极细胞反应的生理学和药理学特性能否在表达TRP通道变体的适当组合的转染HEK细胞中重现？本研究的数据将有助于阐明ON双极细胞中的信号通路，这是视觉处理中的一个基本但知之甚少的步骤。 公共卫生相关性：先天性静止性夜盲症（Congenital Stationary Night Blindness，CSNB）是一种遗传性眼部疾病，可导致夜盲症，也常导致近视、眼球震颤（眼球不自主运动）和视力下降，即使在正常照明条件下也是如此。这项拟议中的研究将确定眼睛中异常的离子通道功能是否是CSNB的原因。

DESCRIPTION (provided by applicant): Congenital stationary night blindness (CSNB) is a group of non-progressive retinal diseases characterized by impaired scotopic vision. Mutations in a number of genes have been shown to be associated with CSNB. They generally affect synaptic transmission between photoreceptors and the second order bipolar cells. When the mutant gene is expressed only in rods, or rod bipolar cells, the phenotype is limited to night blindness. However, when the mutant gene function is also required for synaptic transmission between cones and cone bipolar cells, further visual symptoms are apparent such as myopia, hyperopia, nystagmus, and reduced visual acuity. One such example are mutations in GRM6, the gene encoding mGluR6, which cause an autosomal recessive form of CSNB. In the retina, visual information is segregated into pathways that respond to increases and decreases in light intensity. At the first retinal synapse, the tonic release of glutamate from photoreceptor terminals maintains a high synaptic concentration in darkness that decreases in response to light. Two types of postsynaptic cells, the ON- and OFF-bipolar cells, respond with opposite polarity to glutamate released by photoreceptors, thus establishing the opposing visual pathways. The basis of signaling in OFF-bipolar cells, which relies on the activation of ionotropic glutamate receptors, is well understood. The signaling pathway that generates the light response in ON-bipolar cells, however, is more complex, and the molecular mechanisms remain to be elucidated. The ON-bipolar cell signaling pathway originates with a unique metabotropic glutamate receptor, mGluR6, which is found on the dendrites of ON-bipolar cells. mGluR6 acts via a G-protein, Go, to regulate the activity of an unidentified cation channel such that the light-induced decrease in synaptic glutamate opens the channel and depolarizes the cell. Recently, it has been reported that CSNB in Appaloosa horses is associated with a mutation causing a reduced expression of the TRPM1 cation channel. We hypothesize that TRPM1, and possibly other related TRP channels, are the cation channels coupled to mGluR6 that mediate the depolarizing light response of ON-bipolar cells. We further suggest that mutations in TRP channels will cause CSNB. Using a combination of biochemical, immunohistochemical, and electrophysiological approaches, we will test this hypothesis by answering the following questions: 1. Which TRP channel variants are expressed in ON-bipolar cells? 2. Do mice that carry null mutations in TRP channels expressed in bipolar cells have CSNB? 3. Can the physiological and pharmacological properties of retinal ON bipolar cell responses be reproduced in transfected HEK cells expressing the proper combination of TRP channel variants? The data from this study will contribute to the elucidation of the signaling pathway in the ON-bipolar cell, a fundamental, yet poorly understood, step in visual processing. PUBLIC HEALTH RELEVANCE: Congenital stationary night blindness (CSNB) is an inherited eye disorder causing night blindness, and also often shortsightedness, nystagmus (involuntary eye movement), and reduced visual acuity, even under normal lighting conditions. The proposed research will determine if abnormal ion channel function in the eye can be a cause of CSNB.