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来维持透明度和组织稳态。在透镜纤维细胞中已鉴定出两种不同的间隙连接蛋白，连接蛋白46（Cx48）和连接蛋白50（Cx 50）。本研究计画的总体目标是了解日本人连接通讯在透镜中的角色。详细了解透镜中间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透镜内稳态以及白内障如何发生，那么对透镜中间隙连接蛋白的功能特性的详细了解是重要的。有三个具体目标：1）研究克隆的透镜缝隙连接蛋白的生物物理特性，定位影响差距连接通道通透性和门控的分子决定因子。我们将调查Cx43，Cx 50和Cx46间隙连接通道的选择性差异的分子基础，通过表达野生型和突变连接蛋白结构在非洲爪蟾卵母细胞和转染细胞系。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的N2 A细胞中缝隙连接通道活性的影响。在一系列平行实验中，我们将使用免疫细胞化学技术来确定转染细胞中野生型和突变型连接蛋白的表达模式。3)探讨半缝隙连接通道在透镜中的生理和病理生理作用。为了确定分化中的纤维细胞是否表达具有半缝隙连接通道特性的通道，我们将使用电生理学技术检查新鲜分离的、新分化的小鼠纤维细胞中的宏观电流。我们将对来自纯合子Cx46（-/-）和Cx 50（-/-）小鼠的分离的、分化的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.