Permeability of Lens Gap Junction Channels

项目摘要 由Cx46和Cx50间隙连接通道介导的细胞间通讯 这对于将离子输送到透镜中以及保持透镜的透明度至关重要。 对KO小鼠以及Cx46和Cx50突变的研究已经确定， 连接偶联导致白内障。本建议的主要重点是审查 缝隙连接（GJ）通道在年龄相关性核性白内障的发展。一个关键因素 影响核性白内障形成的因素是， 透镜核中的还原型谷胱甘肽（GSH）。GSH水平的这种降低在 外皮层，因此已被归因于形成一个屏障的扩散， GSH随着年龄的增长而增加。我们已经用电生理技术证明， Cx46和Cx50通道对GSH是可渗透的。进一步的研究表明，GSH可能是 通过Cx46 GJ从合成抗氧化剂的外皮层细胞扩散， 到透镜核中代谢静止的细胞。在本提案中，我们将研究 GJ通道在：（1）在透镜核中建立还原环境，和（2） 介导随着老化而发生的向氧化环境的转变。我们将使用成像技术， 直接测量谷胱甘肽氧化还原电位变化的质谱法 在完整的透镜中从透镜周边到中心。在目标1中，我们将评估 耦合水平对完整透镜中谷胱甘肽氧化还原电位的空间分布的影响。在目标2中， 将决定缝隙连接对其他代谢物的渗透性，这些代谢物是 透镜深处细胞的整体氧化还原状态。在目标3中，我们将研究 GJ通道功能和谷胱甘肽氧化还原电位的变化之间的关系， 年龄具体来说，我们将确定Cx46和Cx50的翻译后修饰是否 随着年龄的增长而积累，导致GJ耦合减少，并最终导致 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通道在保持透镜透明度中的关键作用。

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.