Signaling mechanisms of retinal bipolar cells

项目摘要/摘要 在视网膜中，视觉信息很快被分成不同的通路，这些通路对视网膜的增减做出反应 光照强度。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，对光的反应迅速减少。两种类型的 突触后细胞，即开双极细胞和离双极细胞(BPC)，以相反的极性响应谷氨酸的释放 通过光感受器，从而建立了在整个生命周期中保持的相反的视觉通路 视觉系统。Off-BPC的信号基础，依赖于离子型谷氨酸的激活 受体，是众所周知的；然而，在ON-BPC中产生光反应的信号通路是 更复杂，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，该受体仅在树突上发现 On-BPC。MGluR6通过G蛋白GO调节未知阳离子通道的活性 光诱导的谷氨酸减少打开了通道，并使细胞去极化。在很多方面，这 事件的顺序类似于光感受器外节段的信号转导通路， 其中视紫红质的光激发通过G蛋白、转导蛋白偶联到cGMP门控的闭合 阳离子通道。在外部部分，光响应的动力学在很大程度上取决于 激活的换能器。当转导蛋白α对GTP进行水解时，转导蛋白失活 亚基，该反应通过与G？5-RGS9-R9AP络合物的相互作用而加速。基因突变 编码这三种蛋白质中任何一种的基因会减缓闪光后的恢复，从而严重损害视力。 我们在ON-BPC树枝晶中发现了两个类似的配合物，G？5-RGS7和G？5-RGS11，这表明 与On-BPC信号转导通路失活的机制相似。我们假设RGS- G？5复合体是ON-BPC树突状细胞中mGluR6信号转导通路的重要组成部分。 结合使用生化、免疫组织化学和电生理方法，我们将测试 这一假说回答了以下问题：1.RGS-G？5复合体是如何形成对 On-BPC到光？2.这些RGS复合体是如何锚定在On-BPC树突中的？ 影响它们的功能？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ON-双极细胞的信号通路 视觉处理的基本步骤，但人们对此知之甚少。

Project Summary/Abstract 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.