REGULATION OF RETINAL GAP JUNCTIONS

该项目的长期目标是确定形成视网膜间隙连接的蛋白质，并了解其调节的分子机制。间隙连接是细胞间通讯的普遍介质，在视觉系统中起着重要作用。大多数类型的视网膜神经元都是通过间隙连接连接的，它们在视觉适应过程中的调制对许多神经元的敏感性和感受野特性产生深远的影响，并影响哺乳动物视杆回路的信号流路径。已知至少有两条通路调节视网膜间隙连接：多巴胺/PKA通路和一氧化氮/PKG通路。为了充分了解视网膜缝隙连接的调控，需要对缝隙连接特性及其调控进行分子表征。我们克隆了两个鲈鱼视网膜间隙连接蛋白，连接蛋白（Cx） 35和34.7，它们定义了连接蛋白基因家族的新分支。这是第一批主要在视网膜和大脑神经元中发现的连接蛋白。我们现在计划研究伽马连接蛋白如何促进视网膜神经元中观察到的间隙连接特性。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分子和生物物理特性，并将它们与视网膜间隙连接的特性联系起来。本提案的具体目标是：(1)识别表达每种连接蛋白的细胞；(2)鉴定和表征γ连接蛋白的哺乳动物同源体；(3)研究密切相关的伽马连接素的渗透率特性差异；(4)表征蛋白激酶对连接蛋白的调控。各种生物化学、生物物理和分子技术将被用来实现这些目标。这些研究将详细分析在视网膜回路中起重要作用的这组关键连接蛋白的调控。该结果将为理解可能导致神经系统疾病的间隙连接耦合缺陷奠定基础。

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.