REGULATION OF RETINAL GAP JUNCTIONS

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媒介，在视觉系统中扮演着重要的角色。大多数类型的视网膜神经元通过缝隙连接，它们在视觉适应过程中的调节对许多神经元的敏感度和感受场特性有深远的影响，并影响哺乳动物视杆细胞环路中的信号流路径。已知至少有两条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。为了充分了解视网膜缝隙连接的调控，需要对缝隙连接的性质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，它们定义了连接蛋白基因家族的新的Gamma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影响在视网膜神经元中观察到的缝隙连接特性。我们将使用的一般策略是在孤立的系统中识别连接蛋白的特征分子和生物物理属性，并将它们与视网膜中缝隙连接的属性联系起来。这一建议的具体目标是：(1)鉴定表达每种连接蛋白的细胞；(2)鉴定和鉴定哺乳动物中与伽玛连接蛋白同源的基因；(3)检测密切相关的伽玛连接蛋白的通透性差异；以及(4)表征蛋白激酶对连接蛋白的调节。将采用各种生化、生物物理和分子技术来实现这些目标。这些研究将提供对这一关键的连接蛋白组的调节的详细分析，连接蛋白在视网膜回路中起着至关重要的作用。这些结果将为理解缝隙连接偶联缺陷奠定基础，缝隙连接偶联缺陷可能导致神经疾病。

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.