Signaling mechanisms of retinal bipolar cells

描述（由申请人提供）：在视网膜中，视觉信息被快速分离到对光强度的增加和减少做出反应的通路中。在第一个视网膜突触处，从光感受器末梢紧张性释放谷氨酸在黑暗中维持高突触浓度，其响应于光而迅速降低。两种类型的突触后细胞，ON-和OFF-双极细胞（BPC），对光感受器释放的谷氨酸产生相反的极性反应，从而建立了相反的视觉通路，并在整个视觉系统的其余部分中维持。依赖于离子型谷氨酸受体的激活的OFF-BPC中的信号传导的基础是很好理解的;然而，在ON-BPC中产生光响应的信号传导途径更复杂，并且分子机制仍有待阐明。ON-BPC信号通路起源于独特的代谢型谷氨酸受体mGluR 6，其仅在ON-BPC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，使得光诱导的谷氨酸减少打开通道并使细胞去极化。在许多方面，这一系列的事件类似于光感受器外节的信号转导途径，其中视紫红质的光激发通过G蛋白转导素偶联到cGMP门控阳离子通道的关闭。在外段中，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。转导素α亚基水解GTP后发生转导素的失活，并且该反应通过与G5-RGS 9-R9 AP复合物的相互作用而加速。编码这三种蛋白质中任何一种的基因突变都会通过减缓闪光后的恢复速度而严重损害视力。我们已经在ON-BPC树突中鉴定了两种类似的复合物，G <$5-RGS 7和G <$5-RGS 11，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复合物是ON-BPC树突中mGluR 6信号转导途径的关键组分。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。RGS-G <$5复合物如何塑造ON-BPC对光的反应？2.这些RGS复合物是如何锚定在ON-BPC树突上的，这又是如何影响它们的功能的？3. mGluR 6通路中还有哪些蛋白质与G <$5-RGS 7和G <$5-RGS 11相互作用？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：拟议的研究产生视网膜ON双极细胞的光反应的细胞内生化途径是相关的治疗失明的发展，包括视觉假体和基因治疗的设计。此外，由于G蛋白偶联受体和途径是大多数药物的靶点，因此这项研究将对许多神经和心血管疾病的治疗干预措施的开发具有广泛的相关性。

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.