REGULATION OF RETINAL GAP JUNCTIONS

视网膜间隙连接的调节

• 批准号: 6723702
• 负责人: JOHN O'BRIEN
• 金额: $ 18.46万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723702
• 关键词: HeLa cells; Osteichthyes; SDS polyacrylamide gel electrophoresis; affinity chromatography; confocal scanning microscopy; fluorescence microscopy; fluorescent dye /probe; gap junctions; gene expression; immunocytochemistry; in situ hybridization; laboratory rabbit; membrane channels; molecular cloning; northern blottings; nucleic acid probes; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein kinase; protein structure function; retinal bipolar neuron; site directed mutagenesis; southern blotting; tissue /cell culture; transcription factor; western blottings

The long term objectives of this project are to identify the proteins that form retinal gap junctions and to understand the molecular mechanisms of their regulation. Gap junctions, ubiquitous mediators of intercellular communication, play a prominent role in the visual system. Most types of retinal neurons are connected by gap junctions and their modulation during visual adaptation has profound effects on sensitivity and receptive field properties of many neurons and influences the path of signal flow in the mammalian rod circuit. At least two pathways are known to modulate certain retinal gap junctions: a dopamine/PKA and a nitric oxide/PKG pathway. In order to understand fully the regulation of retinal gap junctions, molecular characterization of the gap junction properties and their modulation is needed. We have cloned two perch retinal gap junction proteins, connexins (Cx) 35 and 34.7, which have defined the new gamma branch of the connexin gene family. These are the first connexins to be found primarily in retinal and brain neurons. We now plan to study how the gamma connexins contribute to the gap junctional properties observed in retinal neurons. The general strategy we will use is to identify characteristic molecular and biophysical properties of the connexins in isolated systems and relate them to properties of the gap junctions in the retina. The specific objectives of this proposal are (1) to identify the cells expressing each connexin; (2) to identify and characterize mammalian homologous of the gamma connexins; (3) to examine the differences in permeability properties of the closely related gamma connexins; and (4) to characterize the regulation of the connexins by protein kinases. A variety of biochemical, biophysical, and molecular techniques will be employed to achieve these goals. These studies will provide a detailed analysis of the regulation of this critical group of connexins that play a vital role in the retinal circuitry. The results will lay the groundwork for understanding defects in gap junctional coupling that may lead to neurological disorders.

该项目的长期目标是确定形成视网膜缝隙连接的蛋白质，并了解其调节的分子机制。缝隙连接是细胞间通讯的重要介质，在视觉系统中起着重要作用。大多数类型的视网膜神经元通过缝隙连接连接，在视觉适应过程中，缝隙连接的调节对许多神经元的敏感性和感受野特性具有深远的影响，并影响哺乳动物杆回路中的信号流路径。已知至少有两种途径调节某些视网膜缝隙连接：多巴胺/PKA和一氧化氮/PKG途径。为了全面了解视网膜缝隙连接的调控，需要对差距连接的特性及其调控进行分子表征。我们已经克隆了两个鲈鱼视网膜缝隙连接蛋白，连接蛋白（Cx）35和34.7，这已经定义了新的γ分支的连接蛋白基因家族。这是第一个主要在视网膜和大脑神经元中发现的连接蛋白。我们现在计划研究γ连接蛋白如何促进视网膜神经元中观察到的差距连接特性。我们将使用的一般策略是识别孤立系统中连接蛋白的特征性分子和生物物理性质，并将它们与视网膜中差距连接的性质联系起来。本提案的具体目标是（1）鉴定表达每种连接蛋白的细胞;（2）鉴定和表征哺乳动物同源的γ连接蛋白;（3）检查密切相关的γ连接蛋白的渗透性特性差异;（4）表征蛋白激酶对连接蛋白的调节。 将采用各种生物化学、生物物理和分子技术来实现这些目标。这些研究将提供一个详细的分析，调节这一关键组的连接蛋白，在视网膜电路中发挥着至关重要的作用。这些结果将为理解可能导致神经系统疾病的缝隙连接偶联缺陷奠定基础。