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来维持透明度和组织动态平衡。在晶状体纤维细胞中发现了两种不同的缝隙连接蛋白，连接蛋白46(Cx48)和连接蛋白50(CX50)。这项研究建议的总体目标是了解JAP连接通讯在晶状体中的作用。详细了解晶状体中缝隙连接通讯的功能特性。如果我们要了解这些连接蛋白如何促进晶状体内环境平衡以及白内障是如何发生的，详细了解晶状体中缝隙连接蛋白的功能特性是很重要的。研究目的有三个：1)研究克隆的晶状体缝隙连接蛋白的生物物理性质，定位缝隙连接通道通透性和门控的分子决定因素。我们将通过在非洲爪哇卵母细胞和转基因细胞系中表达野生型和突变型连接蛋白结构，来研究Cx43、CX50和Cx46缝隙连接通道对带电分子选择性差异的分子基础。2)研究连接蛋白突变对先天性白内障的影响。我们将使用双全细胞膜片钳技术进一步研究突变对转基因N2A细胞缝隙连接通道活性的影响。在一系列平行的实验中，我们将使用免疫细胞化学技术来确定野生型和突变型连接蛋白在转基因细胞中的表达模式。3)探讨晶状体连接半缝隙连接通道在生理和病理生理条件下的作用。为了确定分化中的纤维细胞是否表达具有半间隙连接通道特性的通道，我们将使用电生理学技术检测新分离的、新分化的小鼠纤维细胞中的宏观电流。我们将对纯合子Cx46(-/-)和CX50(-/-)小鼠分离的分化纤维细胞进行类似的实验，以证明连接蛋白是半通道样电流的基础。此外，我们还将研究半通道激活的几种可能机制。

The lens is an avascular, syncytial organ that is dependent on the intercellular communication for the maintenance of transparency and tissue homeostasis. Two different gap junctional proteins, Connexin 46 (Cx48) and Connexin 50 (Cx50), have been identified in lens fiber cells. The overall objective of this research proposal is to understand the role of jap junctional communication in the lens. Detailed knowledge of the functional properties of gap junctional communication in the lens. Detailed knowledge of the functional properties of gap junctional proteins in the lens is important if we are to understand how these connexins contribute to lens homeostasis and how cataracts arise. There are three specific aims: 1) To study the biophysical properties of cloned lens gap junctional proteins and to localize the molecular determinants of permeability and gating of the gap junctional channels. We will investigate the molecular basis for differences in the selectivity of Cx43, Cx50 and Cx46 gap junctional channels for charged molecules by expressing wild-type and mutant connexin constructs in Xenopus oocytes and transfected cell lines. 2) To examine the effects of connexin mutations associated with congenital cataracts. We will further examine the effects of the mutations on gap junctional channel activity in transfected N2A cells using a dual whole cell patch clamp technique. In a parallel series of experiments, we will use immunocytochemical techniques to determine the pattern of expression of wild-type and mutant connexins in transfected cells. 3) To explore the role of junctional hemi-gap junctional channels in the lens under physiological and pathophysiological conditions. To determine if differentiating fiber cells express channels that have the properties of hemi-gap junctional channels, we will examine macroscopic currents in freshly isolated, newly differentiating mouse fiber cells, using electrophysiological techniques. We will perform similar experiments on isolated, differentiating fiber cells from homozygous Cx46(-/-) and Cx50 (-/-) mice to demonstrate that connexins underlie the hemichannel-like currents. In addition, we will examine several possible mechanisms for hemichannel activation.