REGULATION OF CELL-TO-CELL CHANNELS IN THE LENS

脊椎动物晶状体中的纤维细胞是由异常广泛的 细胞间连接。有人提出，这些交汇点 包含细胞间通道，基于细胞间的演示 晶状体内小分子的运动。渠道的功能 在晶状体中的作用是不被理解的，尽管有人提出它们 为代谢产物的运动和调节提供关键途径 分子。为了深入了解功能方面， 这些渠道将在这里进行检查。最终，我们希望确定 具有生理意义的分子在细胞之间移动 镜头，并了解这些运动的含义。本研究 结合了细胞方法和生化性质的方法。在 以前培养的“晶状体”将被检查，因为它们是极好的 晶状体纤维细胞模型。荧光染料在不同染料之间的转移 将对细胞进行定量监测，以检测 细胞到细胞的通道。下面将分析渗透率的变化 变化：a)细胞内pH和钙水平，b)cAMP水平 和相关蛋白激酶的活性，c)在蛋白质活性中 激酶C和D)与细胞损伤相关的方面。学习与研究 蛋白激酶与体内表观晶状体的磷酸化有关 结合蛋白，MP26。协作性全细胞膜片钳实验 以探索连接通道的各种属性。 膜囊泡将被用来在更多的时间内清除通道调节 定义的系统。首先将对囊泡进行表征，以便 了解MP26的形貌和制备条件 影响渠道活跃度。因为细胞到细胞的全部活动 可能找不到“半经”形式的经络，我们也会开始 开发一种用于组装完整的细胞到细胞通道的测试。 我们相信，这些研究将：a)增进了解 细胞间通道在晶状体中的作用，以及b)在 对晶状体中不同疾病状态的解释(例如，在 某些白内障的接合处可能不提供缓冲 细胞差异)。

The fiber cells in the vertebrate lens are connected by unusually extensive intercellular junctions. It has been suggested that these junctions contain cell-to-cell channels, based on demonstrations of the intercellular movement of small molecules within the lens. The functions of the channels in the lens are not understood, although it has been proposed that they provide critical pathways for the movement of metabolites and regulatory molecules. To gain insights into the functional aspects, the regulation of the channels will be examined here. Ultimately, we hope to identify the molecules of physiological significance that move between cells within the lens and to understand the implications of these movements. This study combines a cellular approach with one of a biochemical nature. In the former, cultured "lentoids" will be examined since they serve as excellent models for lens fiber cells. The transfer of fluorescent dyes between cells will be monitored in a quantitative manner in order to assay for cell-to-cell channels. Changes in permeability will be analyzed following alterations: a) in intracellular pH and calcium levels, b) in cAMP levels and the activity of related protein kinases, c) in the activity of protein kinase C and d) in aspects related to cell injury. Studies with the kinases relate to the in vivo phosphorylation of the apparent lens junctional protein, MP26. Collaborative whole-cell patch-clamp experiments are included to explore various properties of the junctional channels. Membrane vesicles will be utilized to pursure channel regulation in a more defined system. The vesicles will first be characterized in order to understand the topography of MP26 and preparative conditions which influence channel activity. Since full activity for the cell-to-cell channels may not be found in the "hemi-channel" form, we will also begin developing an assay for the assembly of a complete cell-to-cell channel. We believe that these studies will: a) lead to an improved understanding of the role of cell-to-cell channels in the lens and b) aid in the interpretation of different disease states in the lens (e.g., in the case of certain cataracts where junctions may not provide for a buffering of cellular differences).