Regulation of retinal gap junctions

描述(申请人提供)：这项研究的长期目标是确定调节视网膜电耦合的机制，并确定哪些调节模式对观察到的不同电耦合神经回路的适应过程最重要。由缝隙连接介导的电耦合参与了大多数类型视网膜神经元的信号处理功能。视觉适应过程中缝隙连接的调制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。在视网膜内的不同回路中，对由相同连接蛋白组成的缝隙连接的需求非常不同，观察到的调节反应也非常不同。以前的结果表明，有几种不同的机制来调节偶联，我们假设这些机制的贡献将根据细胞类型的不同而不同。这个项目的重点是描述由Cx35/36组成的缝隙连接的主要调控机制，Cx35/36是视网膜神经元和整个中枢神经系统中分布最广泛的连接蛋白。本项目要检验的具体假设是：1)特定残基的磷酸化状态表征了Cx35缝隙连接的调节状态；2)钙调蛋白参与了缝隙连接偶联的钙依赖调节；3)偶联的调节受到与Cx35密切相关的复合体中蛋白质的影响。各种体外生物物理和生化技术将被用来表征Cx35的翻译后修饰和蛋白质结合相互作用。破坏这些过程的突变将在细胞培养表达系统中进行，并研究它们对调控的影响。免疫染色将检测调节通路的组成部分与视网膜神经元中Cx35的关联。这些研究将确定在影响缝隙连接耦合的视网膜疾病中可能受到不当调控的主要部位。

DESCRIPTION (provided by applicant): The long-term objectives of this study are to identify the mechanisms that regulate electrical coupling in the retina, and to determine which modes of regulation are most important for the adaptive processes observed n different electrically coupled neural circuits. Electrical coupling, mediated by gap junctions, contributes to the signal processing functions of most types of retinal neurons. Modulation of gap junctions 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. In different circuits within the retina, very different demands are placed on gap junctions made of the same connexin proteins, and the regulatory responses observed are quite disparate. Previous results suggest that there are several different mechanisms that regulate coupling, and we hypothesize that the contributions of these will differ depending on the cell type. This project focuses on delineating the major mechanisms that regulate gap junctions made of Cx35/36, the most widespread connexin in retinal neurons and throughout the central nervous system. The specific hypotheses to be examined in this project are that: 1) the phosphorylation state of specific residues characterizes the regulatory state of Cx35 gap junctions; 2) calmodulin is involved in calcium-dependent regulation of gap junction coupling; and 3) regulation of coupling is influenced by proteins closely associated with Cx35 in a complex. A variety of in vitro biophysical and biochemical techniques will be used to characterize posttranslational modifications and protein binding interactions of Cx35. Mutations that disrupt these processes will be made and their effects on regulation studied in cell culture expression systems. The association of components of the regulatory pathways with Cx35 in retinal neurons will be examined by immunostaining. These studies will identify the major sites that could be subject to improper regulation in retinal disorders that affect gap junction coupling.