Intercellular Communication in the Lens

晶状体是一种无血管的合胞器官，依赖于细胞间的交流来维持透明度和组织稳态。在晶状体纤维细胞中发现了两种不同的间隙连接蛋白，Connexin 46 （Cx48）和Connexin 50 （Cx50）。本研究计划的总体目标是了解晶状体中japan junction communication的作用。详细了解晶体间隙结通信的功能特性。如果我们要了解晶状体间隙连接蛋白如何促进晶状体稳态和白内障的发生，那么详细了解晶状体间隙连接蛋白的功能特性是很重要的。本研究的主要目的有三个：1)研究克隆晶状体间隙连接蛋白的生物物理特性，定位晶状体间隙连接通道通透性和门控性的分子决定因素。我们将通过在非洲爪蟾卵母细胞和转染细胞系中表达野生型和突变型连接蛋白，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)探讨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染N2A细胞间隙连接通道活性的影响。在平行的一系列实验中，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染细胞中的表达模式。3)探讨晶状体连接半间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，我们将使用电生理技术检查新分离的、新分化的小鼠纤维细胞中的宏观电流。我们将在纯合子Cx46（-/-）和Cx50（-/-）小鼠的分离分化纤维细胞上进行类似的实验，以证明连接蛋白是半通道样电流的基础。此外，我们将研究几种可能的半通道激活机制。

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.