TRP channel expression and function in ON-bipolar cells

描述(申请人提供)：先天性静止性夜盲(CSNB)是一组以暗视损害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第二级双极细胞之间的突触传递。当突变基因仅在杆状细胞或杆状双极细胞中表达时，表型仅限于夜盲症。然而，当突变的基因功能也需要在视锥细胞和视锥双极细胞之间进行突触传递时，进一步的视觉症状就会明显，如近视、远视、眼球震颤和视力下降。一个这样的例子是编码mGluR6的基因GRM6的突变，它导致了常染色体隐性形式的CSNB。在视网膜中，视觉信息被分成不同的路径，这些路径对光强度的增加和减少做出反应。在第一次视网膜突触，谷氨酸从光感受器终端的紧张性释放在黑暗中保持较高的突触浓度，对光的反应减少。两种类型的突触后细胞，即开双极细胞和离双极细胞，以相反的极性对光感受器释放的谷氨酸做出反应，从而建立相反的视觉通路。非双极细胞的信号基础依赖于离子型谷氨酸受体的激活，这一点是众所周知的。然而，在双极细胞中产生光响应的信号通路更加复杂，其分子机制仍有待阐明。ON-双极细胞信号通路起源于一种独特的代谢性谷氨酸受体mGluR6，它存在于ON-双极细胞的树突上。MGluR6通过G蛋白GO调节一种未知阳离子通道的活性，从而光诱导突触谷氨酸的减少打开该通道并使细胞去极化。最近，有报道称，Appaloosa马的CSNB与导致TRPM1阳离子通道表达减少的突变有关。我们假设TRPM1和其他相关的Trp通道是连接到mGluR6的阳离子通道，介导双极细胞的去偏振光响应。我们进一步认为Trp通道的突变将导致CSNB。我们将结合生化、免疫组织化学和电生理学的方法，通过回答以下问题来验证这一假说：1.在双极细胞中表达哪些Trp通道变体？2.在双极细胞中表达Trp通道零突变的小鼠是否存在CSNB？3.在表达Trp通道变体正确组合的HEK细胞中，能否复制视网膜对双极细胞的生理和药理学反应？这项研究的数据将有助于阐明双极细胞的信号通路，这是视觉处理中的一个基本步骤，但人们对此知之甚少。 与公共卫生相关：先天性静止性夜盲(CSNB)是一种遗传性眼病，会导致夜盲，即使在正常的照明条件下，也经常会出现近视、眼球震颤(非自主眼球运动)和视力下降。这项拟议的研究将确定眼睛离子通道功能异常是否可能是CSNB的原因之一。

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.