REGULATION OF CELL-TO-CELL CHANNELS IN THE LENS

脊椎动物透镜中的纤维细胞通过异常广泛的连接 细胞间连接 有人认为这些连接点 包含细胞间通道，基于细胞间 小分子在透镜内的运动。 渠道的功能 在透镜中，尽管已经提出它们 为代谢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， 这里将检查通道。 最后，我们希望能找出 具有生理意义的分子，在细胞内的细胞之间移动， 透镜并理解这些运动的含义。 本研究 结合了细胞学方法和生物化学方法。 在 前，培养的“lentoids”将被检查，因为他们作为优秀的 透镜纤维细胞的模型。 荧光染料在 将以定量方式监测细胞，以测定 细胞间的通道 渗透率的变化将在以下进行分析 改变：a）细胞内pH和钙水平，B）cAMP水平 和相关蛋白激酶的活性，c）在蛋白激酶的活性中， 激酶C和d）在与细胞损伤相关的方面。 研究与 与表观透镜的体内磷酸化有关的激酶 连接蛋白MP26。 协同全细胞膜片钳实验 以探索连接通道的各种特性。 膜囊泡将被用来在更大范围内进行通道调节 定义系统。 囊泡将首先被表征，以便 了解MP26的形貌和制备条件， 影响渠道活动。 由于细胞间的全面活动 通道可能不会在“半通道”形式中找到，我们也将开始 开发用于组装完整的细胞-细胞通道的测定。 我们相信，这些研究将：a）导致更好的理解 细胞间通道在透镜中的作用，和B）有助于 对透镜中不同疾病状态的解释（例如，的情况下 某些白内障的交界处可能无法提供缓冲， 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).