Intercellular Communication in the Lens

晶状体是一种无血管的合体器官，依赖于细胞间的通讯 保持透明度和组织动态平衡。三种不同的连接蛋白(连接蛋白 43(Cx43)、连接蛋白46(Cx46)、连接蛋白50(CX50))存在于晶状体纤维中。整体而言 这项研究的目的是为了更好地了解缝隙连接蛋白在晶状体中的作用。这个 将被检验的中心假说是连接蛋白缝隙连接和半突触都扮演着 在保持镜头透明度方面起着重要作用。有两个具体目标： 目的1.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CX50的缝隙连接通道和半通道。我们将继续 研究人晶状体连接蛋白野生型和突变型表达的功能特性 异种细胞系统。将要研究的两种缝隙连接蛋白特性是：(1) 分子对较大渗透剂的渗透性；(2)半通道门。此外，功能和 几种与先天性白内障相关的连接蛋白基因突变对细胞的影响 将测试终端和E1域。 目的2.确定纤维细胞中非选择性漏电导的分子同一性 通过去除外部钙而激活的。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生产出一种可行的、分离的纤维细胞制剂。我们将使用 该制剂以及转染不同晶状体连接蛋白的HeLa细胞 下列问题：被激活的非选择性漏电导的分子同一性是什么 去除外部钙？这种电导能解释发生在的钠的内流吗 生理条件下的纤维细胞？这种电导是否调节病理性钙离子？ 纤维细胞因细胞肿胀而产生的内流？需要检验的假设是 连接蛋白半通道是所有这些现象的原因。我们还将使用孤立的 制备纤维细胞以研究多种因素对缝隙连接电导的调节 以前被证明在调节细胞间通讯方面起着重要作用 完整的晶状体，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.