Regulation of retinal gap junctions

描述（由申请人提供）：本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在不同电耦合神经回路中观察到的适应性过程最重要。由缝隙连接介导的电耦合有助于大多数类型的视网膜神经元的信号处理功能。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，并影响哺乳动物视杆细胞回路中的信号流路径。在视网膜内的不同回路中，对由相同连接蛋白组成的间隙连接的要求非常不同，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耦合，我们假设这些机制的贡献将取决于细胞类型。该项目的重点是描绘调节缝隙连接的主要机制，缝隙连接由Cx 35/36组成，Cx 35/36是视网膜神经元和整个中枢神经系统中最广泛的连接蛋白。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Cx 35间隙连接的调节状态; 2）钙调蛋白参与间隙连接偶联的钙依赖性调节; 3）偶联的调节受到复合体中与Cx 35密切相关的蛋白质的影响。各种体外生物物理和生物化学技术将用于表征Cx 35的翻译后修饰和蛋白结合相互作用。将进行破坏这些过程的突变，并在细胞培养表达系统中研究其对调控的影响。将通过免疫染色检查视网膜神经元中调节途径的组分与Cx 35的关联。这些研究将确定在影响缝隙连接偶联的视网膜疾病中可能受到不适当调节的主要部位。

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.