Intercellular Communication in the Lens

描述(申请人提供)：晶状体是一种无血管的合胞器官，依赖于细胞间的通讯来维持透明度和组织内稳态。在晶状体纤维中发现了三种不同的连接蛋白：连接蛋白43(Cx43)、连接蛋白46(Cx46)和连接蛋白50(CX50)。这项研究计划的总体目标是更好地了解缝隙连接蛋白在晶状体中的作用。将被检验的中心假设是，连接蛋白缝隙连接和半通道在维持晶状体透明度方面都发挥着重要作用。目的1.了解野生型和突变型Cx46和CX50缝隙连接通道和半管的N端、第一跨膜跨膜区(M1)和第一胞外区(E1)的结构差异是如何导致其生物物理性质差异的。我们将继续研究在异源细胞系统中表达的野生型和突变型晶状体连接蛋白的功能特性。将要研究的两个连接蛋白特性是：(1)分子对较大渗透剂的渗透性；(2)半通道门控。此外，还将测试几种与先天性白内障相关的连接蛋白突变对功能和细胞的影响，这些突变定位于N-末端和E1域。目的2.确定外源性钙离子清除激活的纤维细胞非选择性渗漏电导的分子同一性。最近，我的实验室开发了一种技术，可以从成年和新生小鼠的晶状体中生产出可行的、分离的纤维细胞制剂。我们将使用这种制剂以及用不同晶状体连接蛋白转染的HeLa细胞来解决以下问题：通过去除外部钙激活的非选择性泄漏电导的分子同一性是什么？这种电导是否可以解释生理条件下纤维细胞中发生的钠内流？这种电导是否介导了纤维细胞对细胞肿胀的病理性钙内流？需要检验的假设是，连接蛋白半通道对所有这些现象负有责任。我们还将使用分离的纤维细胞准备来研究各种因素对缝隙连接电导的调节，这些因素在调节完整晶状体的细胞间通讯方面发挥着重要作用，如磷酸化、氧化应激和PHI。公共卫生相关性这项研究的总体目标是了解缝隙连接蛋白在晶状体中的作用。了解缝隙连接蛋白的功能特性将有助于更好地理解连接蛋白如何促进晶状体内稳态以及白内障是如何发生的。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE The overall goal of this research is to understand the role of gap junctional proteins in the lens. Understanding the functional properties of gap junctional proteins will lead to a better understanding of how connexins contribute to lens homeostasis and how cataracts arise.