Regulation of retinal gap junctions

这项研究的长期目标是确定调节电耦合的机制， 视网膜，并确定哪些模式的监管是最重要的自适应过程中观察到的 n个不同的电耦合神经回路。由间隙连接介导的电耦合有助于 大多数视网膜神经元的信号处理功能。视神经细胞间隙连接的调节 适应对许多神经元的敏感性和感受野特性有深刻的影响， 哺乳动物视杆细胞回路中的信号流路径。在视网膜的不同回路中， 对由相同连接蛋白组成的间隙连接的需求，以及调节反应， 观察到的是完全不同的。以前的结果表明，有几种不同的机制， 调节偶联，我们假设这些的贡献将根据细胞类型而不同。 本项目的重点是阐明调控缝隙连接的主要机制，缝隙连接由Cx 35/36组成， 在视网膜神经元和整个中枢神经系统中分布最广的连接蛋白。具体 本研究的假设是：1）特定残基的磷酸化状态 2）钙调蛋白参与钙依赖性的细胞凋亡; 间隙连接偶联的调节;和3）偶联的调节受密切相关的蛋白质的影响 与Cx 35在一个复杂的。将使用各种体外生物物理和生物化学技术， 表征Cx 35的翻译后修饰和蛋白结合相互作用。突变会破坏 将进行这些过程，并在细胞培养表达系统中研究它们对调节的影响。的 视网膜神经元中调节途径的组分与Cx 35的关联将通过以下方法来检查： 免疫染色。这些研究将确定可能受到不当监管的主要地点， 影响缝隙连接耦合的视网膜疾病。

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.