Signaling mechanisms of retinal bipolar cells

描述(申请人提供)：在视网膜中，视觉信息被迅速分离成对光强度的增加和减少做出反应的通路。在第一个视网膜突触，谷氨酸从光感受器终端的紧张性释放在黑暗中保持着较高的突触浓度，对光的反应迅速下降。两种类型的突触后细胞，即开双极细胞和离双极细胞(BPC)，对光感受器释放的谷氨酸做出相反的极性反应，从而建立起贯穿视觉系统其余部分的相反的视觉通路。Off-BPC的信号基础依赖于离子型谷氨酸受体的激活，这是众所周知的；然而，On-BPC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-BPC信号通路起源于一种独特的代谢性谷氨酸受体mGluR6，该受体仅存在于On-BPC的树突上。MGluR6通过一种名为GO的G蛋白来调节一种未知阳离子通道的活性，从而使光诱导的谷氨酸减少打开该通道并使细胞去极化。在许多方面，这一事件序列类似于光感受器外节的信号转导通路，其中视紫红质的光激发通过G蛋白转导蛋白偶联到cGMP门控阳离子通道的关闭。在外段，光响应的动力学在很大程度上取决于激活的转导蛋白的寿命。当转导蛋白α亚基对GTP进行水解时，转导蛋白失活，这一反应通过与G？5-RGS9-R9AP复合体的相互作用而加速。编码这三种蛋白质中任何一种的基因突变会减缓闪光后的恢复，从而严重损害视力。我们在ON-BPC树突中发现了两个类似的复合体，G？5-RGS7和G？5-RGS11，这表明ON-BPC信号转导通路的失活机制类似。我们假设RGS-G？5复合体是ON-BPC树突状细胞中mGluR6信号转导通路的关键成分。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.RGS-G？5复合体如何影响ON-BPC对光的反应？2.这些RGS复合体是如何锚定在ON-BPC树突中的，这对它们的功能有什么影响？3.mGluR6途径中还有哪些蛋白质与G？5-RGS7和G？5-RGS11相互作用？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。公共卫生相关性：对产生视网膜双极细胞光反应的细胞内生化途径的拟议研究与失明治疗方法的开发有关，包括视觉假体的设计和基因治疗。此外，由于G蛋白偶联受体和通路是大多数药物的靶点，本研究将对开发多种神经和心血管疾病的治疗干预措施具有广泛的相关性。

DESCRIPTION (provided by applicant): In the retina, visual information is quickly 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 rapidly decreases in response to light. Two types of postsynaptic cells, the ON- and OFF-bipolar cells (BPCs), respond with opposite polarity to glutamate released by photoreceptors, thus establishing the opposing visual pathways that are maintained throughout the rest of the visual system. The basis of signaling in OFF-BPCs, which relies on the activation of ionotropic glutamate receptors, is well understood; the signaling pathway that generates the light response in ON-BPCs, however, is more complex, and the molecular mechanisms remain to be elucidated. The ON-BPC signaling pathway originates with a unique metabotropic glutamate receptor, mGluR6, which is found exclusively on the dendrites of ON-BPCs. mGluR6 acts via a G-protein, Go, to regulate the activity of an unidentified cation channel such that the light-induced decrease in glutamate opens the channel and depolarizes the cell. In many ways, this sequence of events resembles the well-studied signal transduction pathway of photoreceptor outer segments, in which photoexcitation of rhodopsin is coupled via the G-protein, transducin, to the closure of a cGMP-gated cation channel. In the outer segment, the kinetics of the light response is largely determined by the lifetime of activated transducin. Deactivation of transducin occurs upon hydrolysis of GTP by the transducin alpha subunit, and this reaction is accelerated by interaction with the G¿5-RGS9-R9AP complex. Mutations in the gene encoding any one of these three proteins severely impair vision by slowing recovery after light flashes. We have identified two similar complexes, G¿5-RGS7 and G¿5-RGS11, in ON-BPC dendrites suggesting a similar mechanism of deactivation of the ON-BPC signal transduction pathway. We hypothesize that the RGS-G¿5 complexes are critical components of the mGluR6 signal transduction pathway in ON-BPC dendrites. Using a combination of biochemical, immunohistochemical, and electrophysiological approaches, we will test this hypothesis by answering the following questions: 1. How do RGS-G¿5 complexes shape the response of ON-BPCs to light? 2. How are these RGS complexes anchored in the ON-BPC dendrites and how does this affect their function? 3. What other proteins in the mGluR6 pathway interact with G¿5-RGS7 and G¿5-RGS11? 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: The proposed research into the intracellular biochemical pathways generating the light response of retinal ON-bipolar cells is of relevance to the development of cures for blindness, including the design of visual prosthetics and gene therapy. Moreover, because G protein-coupled receptors and pathways are the target of the majority of pharmaceutical drugs, this research will have broad relevance to the development of therapeutic interventions for numerous neurological and cardiovascular diseases.