Intercellular Communication in the Lens

透镜是一种无血管的合胞体器官，其依赖于细胞间通讯以进行细胞间通讯。 保持透明度和组织稳态。三种不同的连接蛋白（Connexin 连接蛋白43（Cx43）、连接蛋白46（Cx46）、连接蛋白50（Cx 50））。整体 本研究计划的目的是更好地了解连接蛋白在透镜中的作用。的 将要检验的中心假设是，连接蛋白间隙连接和半通道都发挥了作用， 保持透镜透明度的重要作用。有两个具体目标： 目标1.为了了解N-末端的结构差异，第一次跨膜 结构域（M1）和第一胞外结构域（E1）导致生物物理性质的差异 野生型和突变型Cx46和Cx 50间隙连接通道和半通道。我们将继续 研究野生型和突变型透镜连接蛋白的功能特性， 异源细胞系统。将研究的两个连接蛋白特异性性质是：（1） 对较大渗透物的分子渗透性;（2）半通道门控。此外，功能和 几种先天性白内障相关连接蛋白突变的细胞效应，这些突变定位于N- 将测试末端和E1结构域。 目标2.为了确定纤维细胞中非选择性漏导的分子身份， 通过去除外部钙而激活。最近，我的实验室开发了一种技术， 从成年和新生小鼠的晶状体中产生活的分离的纤维细胞制剂。我们将使用 该制剂以及用不同的透镜连接蛋白转染的HeLa细胞， 下面的问题：什么是非选择性泄漏电导激活的分子身份 去除外部钙？这种电导能解释发生在 生理条件下的纤维细胞？这种电导是否介导了病理性钙离子 纤维细胞中因细胞肿胀而发生的流入？有待检验的假设是， 连接蛋白半通道负责所有这些现象。我们还将使用隔离的 纤维细胞制备，以研究各种因素对缝隙连接电导的调节， 先前已显示在调节细胞间通讯中起重要作用， 完整透镜如磷酸化、氧化应激和pHi。

The lens is an avascular, syncytial organ that is dependent on intercellular communication for the maintenance of transparency and tissue homeostasis. Three different connexin proteins (Connexin 43 (Cx43), Connexin 46 (Cx46), Connexin 50 (Cx50)) have been identified in lens fibers. The overall objective of this research proposal is to better understand the role of connexins in the lens. The central hypothesis that will be tested is that both connexin gap junctions and hemichannels play an important role in maintaining lens transparency. There are two specific aims: Aim 1. To understand how structural differences in the N-terminal, first transmembrane spanning domain (M1), and first extracellular domain (E1) contribute to differences in the biophysical properties of wild-type and mutant Cx46 and Cx50 gap junctional channels and hemichannels. We will continue to investigate the functional properties of wild-type and mutant lens connexins expressed in heterologous cell systems. The two connexin specific properties that will be studied are: (1) molecular permeability to larger permeants; (2) hemichannel gating. In addition, the functional and cellular effects of several congenital cataract associated connexin mutations that localize to the N- terminal and E1 domains will be tested. Aim 2. To determine the molecular identity of the nonselective leak conductance in fiber cells that is activated by removal of external calcium. Recently, my laboratory has developed a technique for producing a viable, isolated fiber cell preparation from lenses of adult and neonatal mice. We will use this preparation as well as HeLa cells transfected with different lens connexins to address the following questions: What is the molecular identity of the nonselective leak conductance activated by removal of external calcium?; Can this conductance account for the influx of sodium that occurs in fiber cells under physiological conditions?; Does this conductance mediate the pathological calcium influx that occurs in fiber cells in response to cell swelling? The hypothesis to be tested is that connexin hemichannels are responsible for all of these phenomena. We will also use the isolated fiber cell preparation to study the regulation of gap junctional conductance by a variety of factors that have been previously shown to play an important role in regulating intercellular communication in the intact lens such as phosphorylation, oxidative stress and pHi.