Permeability of Lens Gap Junction Channels

项目总结 CX46和CX50缝隙连接通道介导的细胞间通讯 它是离子进入晶状体和维持晶状体透明度的中心。 对KO小鼠和Cx46和CX50突变的研究已经证实，GAP的缺失 交界性偶联导致白内障。这项提案的主要焦点是审查 缝隙连接(GJ)通道在老年性核性白内障发生中的作用一个关键因素 影响核性白内障形成的因素是随着年龄的增长而突然下降。 晶状体核中的还原型谷胱甘肽(GSH)。谷胱甘肽水平的这种下降在 外皮质，因此被认为是由于形成了一道屏障，阻止了 随年龄增长的谷胱甘肽。我们已经使用电生理技术来证明 CX46和CX50通道对GSH具有通透性。更多研究表明，谷胱甘肽很可能 通过Cx46 GJS从合成抗氧化剂的外层皮质细胞扩散， 到晶状体核中代谢静止的细胞。在这份提案中，我们将研究 GJ通道：(1)晶状体核内还原环境的建立；(2) 调节向氧化环境的转变，这种环境随着年龄的增长而发生。我们将使用成像和 直接测定谷胱甘肽氧化还原电位变化的质谱法 在完整的晶状体中，从晶状体外围到中心。在目标1中，我们将评估 在完整晶状体中谷胱甘肽氧化还原电位空间分布的耦合水平。在目标2中，我们 将决定缝隙连接对其他已知的代谢产物的渗透性 晶状体深处细胞的整体氧化还原状态。在目标3中，我们将考察两者之间的关系 GJ通道功能和谷胱甘肽氧化还原电位的变化之间的关系 年龄。具体地说，我们将确定对Cx46和CX50的翻译后修改 随着年龄的增长而积累，导致GJ耦合减少，并最终发展为 GSH扩散的障碍。因此，这些研究直接有助于理解 ARN白内障形成的机制，并将进一步强调 GJ通道在维持晶状体透明度方面的关键作用。

PROJECT SUMMARY Intercellular communication mediated by Cx46 and Cx50 gap junction channels is central to the transport of ions into the lens and to the maintenance of lens transparency. Studies on KO mice and on Cx46 and Cx50 mutations have established that the absence of gap junctional coupling leads to cataracts. The main focus of this proposal is to examine the role of gap junction (GJ) channels in the development of age-related nuclear cataracts. A key factor that influences nuclear cataract formation is the abrupt age-dependent decline in the levels of reduced glutathione (GSH) in the lens nucleus. This reduction in GSH levels is not observed in the outer cortex and therefore has been attributed to the formation of a barrier to the diffusion of GSH that develops with age. We have used electrophysiological techniques to show that that Cx46 and Cx50 channels are permeable to GSH. Additional studies indicated that GSH is likely to diffuse via Cx46 GJs from the cells in the outer cortex, where the anti-oxidant is synthesized, to the metabolically quiescent cells in the lens nucleus. In this proposal, we will examine the role of GJ channels in: (1) the establishment of a reducing environment in the lens nucleus, and (2) mediating the shift to an oxidized environment that occurs with aging. We will use imaging and mass spectrometry methods to directly measure the variation in the glutathione redox potential from the lens periphery to the center in the intact lens. In Aim 1, we will assess the influence of coupling levels on the spatial profile of glutathione redox potential in the intact lens. In Aim 2, we will determine the permeability of gap junctions to other metabolites known to be essential for the overall redox state of cells deep in the lens. In Aim 3, we will examine the relationship between GJ channel functionality and changes in the glutathione redox potential that occur with age. Specifically, we will determine whether post-translational modifications to Cx46 and Cx50 accumulate with age, leading to a reduction in GJ coupling, and ultimately to the development of the barrier to GSH diffusion. These studies thus make a direct contribution to the understanding of the mechanisms underlying the formation of ARN cataracts, and will serve to further highlight the key role of GJ channels in maintenance of lens transparency.