Chemical architecture of retinal circuits

视网膜回路的化学结构

• 批准号: 7034510
• 负责人: NOGA VARDI
• 金额: $ 50.39万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7034510
• 关键词: G protein; Xenopus; biological signal transduction; biophysics; calcium; chemical structure; electroretinography; gene targeting; genetic library; genetically modified animals; glutamate receptor; immunocytochemistry; laboratory mouse; membrane proteins; neuropeptides; phosphorylation; polymerase chain reaction; retina; retinal bipolar neuron; voltage gated channel

DESCRIPTION (provided by applicant): My long-term goal is to understand intracellular signaling cascades and their contribution to image processing in retina. As the eye scans a scene, each photoreceptor sees alternating pulses of light and dark that decrease or increase its glutamate release. Each burst of glutamate has two effects: to directly open an AMPA/kainate cation channel in OFF bipolar cells, and to indirectly close an unidentified cation channel in ON bipolar cells. The first step toward channel closure is activation of the G-protein Go1 by the metabotropic receptor mGluR6, but the rest of the cascade is largely unknown. Our goal is to elucidate the full cascade, its regulators, and the channel. We have now identified two strong interactors of Galphao1, Ret-RGS1 and Pcp2. Both colocalize with mGluR6 and Galphao1, coimmunoprecipitate with Galphao1, and modulate G(o1's activity in vitro. Ret-RGS1 catalyzes Galphao's GTPase activity (to deactivate Go), and Pcp2 is thought to inhibit GDP dissociation from Galphao (to slow Go's activation). Because the light response arises when Go is deactivated, and terminates when Go is activated, Ret-RGS1 is hypothesized to accelerate the rising phase of the light response and Pcp2 to prolong its falling phase. To test these hypotheses AIMS 1 and 2 will: (1) test effect of Pcp2 in oocytes expressing the mGluR6 transduction elements; (2) record the dynamics of the electroretinogram b-wave from Pcp2- and ret-RGS1-null mice; and (3) record direct responses from ON bipolar cells of these null mice to puffs of mGluR6 antagonist (whole cell configuration). AIM 3 will identify the ON bipolar transduction channel. This will be accomplished by expression cloning and hybridization using an ON bipolar cDNA library. We have already produced a transgenic mouse with EGFP-expressing ON bipolar cells and have isolated these cells. The isolated cells have been used to construct an ON bipolar cDNA library. The library will be expressed with mGluR6 and G(o1 in Xenopus oocytes, and responses to mGluR6 agonist will be tested. AIM 4 will characterize the channel's fundamental biophysical properties, such as its gating molecules, voltage dependence, sensitivity to Ca2+, and effect of phosphorylation. This effort will contribute fundamental understanding of the first synapse on the visual pathway and of night blindness; it should thus extend the basic foundation for future clinical studies.

描述（由申请人提供）：我的长期目标是了解细胞内信号级联及其对视网膜图像处理的贡献。当眼睛扫描场景时，每个光感受器都会看到光与黑暗的交替脉冲，从而减少或增加其谷氨酸释放。谷氨酸爆发的每一次爆发都有两种效果：直接在双极细胞中直接打开AMPA/海藻酸盐阳离子通道，并间接关闭双极细胞上未识别的阳离子通道。迈向渠道闭合的第一步是代谢受体mglur6激活G蛋白GO1，但级联的其余部分在很大程度上是未知的。我们的目标是阐明完整的级联，其监管机构和频道。现在，我们已经确定了Galphao1，RET-RGS1和PCP2的两个强相互作用。 Both colocalize with mGluR6 and Galphao1, coimmunoprecipitate with Galphao1, and modulate G(o1's activity in vitro. Ret-RGS1 catalyzes Galphao's GTPase activity (to deactivate Go), and Pcp2 is thought to inhibit GDP dissociation from Galphao (to slow Go's activation). Because the light response arises when Go is停用，当GO激活时终止，RET-RGS1被假设，以加速光响应的上升阶段和pcp2延长其下降阶段，以测试这些假设的目标1和2。 RET-RGS1无效的小鼠；（3）在这些无效的小鼠上的直接响应到MGLUR6拮抗剂的泡沫（全细胞构型）。这些细胞。分离的细胞已用于构建双极cDNA文库。该库将用MglUR6和g表示（Xenopus卵母细胞中的O1，对MGLUR6激动剂的响应将进行测试。AIM4将表征该频道的基本生物物理特性，例如其倾斜分子，电压依赖性，对CAC2+的敏感性和临时贡献。因此，它应该为将来的临床研究扩展基础。