LENS METABOLIC COOPERATION GAP JUNCTIONS AND CATARACT

由于其独特的结构和功能，晶状体代谢和离子 体内平衡绝对取决于晶状体纤维保持互连 通过间隙连接通讯途径到达表面上皮细胞。 离子稳态对于避免白内障至关重要：降水 晶状体纤维中高浓度的可溶性蛋白质 细胞质。 该提案的长期目标是 通过实验证明细胞间通讯的功能 晶状体发育和稳态中的间隙连接。 实验是 概述了其设计用于执行连接蛋白的特定块 镜头中的功能。 Cx43 基因敲除小鼠现已上市 商业上。 这些动物在出生时死于心脏缺陷，因此存活下来 足够长的时间来研究发育和稳态 Cx43 敲除的后遗症，Cx43 首先在正常晶状体中表达 发育，并且在上皮细胞和成熟晶状体中持续存在 细胞和分化纤维。 遮挡其他镜头的功能 连接蛋白，反式显性阴性结构，干扰 连接蛋白功能将在小鼠中以转基因的形式表达 αA-晶状体蛋白启动子，将表达靶向晶状体。 在 雏鸡，活性反式显性阴性构建体将被引入 通过逆转录病毒感染发育晶状体，使用复制- 感受态 RCAS-A 禽病毒。 第二个具体目标是磷酸化 连接蛋白上的位点将通过传统的肽图谱进行绘制 测序。 鉴定出关键的磷酸化丝氨酸和苏氨酸残基 通过这种映射将被突变并被标记表位。 这些连接蛋白 突变体将通过转染引入培养的晶状体细胞中并 使用 RCAS 逆转录病毒将其转化为整个镜片。 变异者的能力 连接蛋白组装成通道，被运输到血浆 膜并靶向间隙连接将随后是 培养物和发育中的晶状体中的免疫组织化学。 将这些突变连接蛋白转染至通讯阴性 Neuro2A 神经母细胞瘤细胞系将允许研究单个细胞的变化 通道电导、电压门控和 pH 敏感性产生的 特定磷酸化残基的损失。

Due to its unique structure and function, lens metabolism and ionic homeostasis depend absolutely on the lens fibers remaining interconnected to the surface epithelial cells by gap junctional communication pathways. Ionic homeostasis is essential in order to avoid cataract: precipitation of the high concentration of soluble proteins in the lens fiber cytoplasms. The long-term objectives of this proposal are to experimentally demonstrate the function of intercellular communication via gap junctions in lens development and homeostasis. Experiments are outlined which are designed carry out specific blocks of connexin functions in the lens. A Cx43 knockout mouse is now available commercially. These animals die at birth of cardiac defects thus living long enough to permit a study of the developmental and homeostatic sequellae of a knockout of Cx43, which is expressed first in normal lens development, and which persists in the mature lens in both epithelial cells and differentiating fibers. To block the function of other lens connexins, trans-dominant negative constructs which interfere with connexin function will be expressed in the mouse as transgenes driven by the alphaA-crystallin promoter, to target expression to the lens. In the chick, active trans-dominant negative constructs will be introduced into the developing lens by retroviral infection, using the replication- competent RCAS-A avian virus. In a second specific aim, phosphorylation sites on connexins will be mapped by conventional peptide mapping and sequencing. Key phosphorylated serine and threonine residues identified by this mapping will be mutated and epitope tagged. These connexin mutants will be introduced into lens cells in culture by transfection and into whole lenses using the RCAS retrovirus. The ability of the mutated connexins to assemble into channels, to be transported to the plasma membrane and to target to gap junctions will be followed by immunohistochemistry in both the cultures and in developing lenses. Transfection of these mutant connexins into the communication-negative Neuro2A neuroblastoma cell line will permit the study of changes in single channel conductance, voltage gating, and pH sensitivity resulting from the loss of specific phosphorylated residues.