LENS METABOLIC COOPERATION GAP JUNCTIONS AND CATARACT

由于其独特的结构和功能，晶状体的新陈代谢和离子 动态平衡完全取决于晶状体纤维的相互连接。 通过缝隙连接通讯通路到达表面上皮细胞。 离子动态平衡是避免白内障的关键：沉淀 晶状体纤维中含有高浓度的可溶性蛋白质 细胞质。这项建议的长期目标是 通过实验演示了细胞间通信的功能 晶状体发育和内稳态中的缝隙连接。实验是 概述了哪些设计用于执行特定的连接蛋白块 在晶状体中起作用。Cx43基因敲除鼠标现已面市 在商业上。这些动物出生时就死于心脏缺陷，因此活了下来 足够长的时间来研究发育和动态平衡 Cx43基因敲除的后遗症，该基因在正常晶状体中首次表达 发育，并持续在成熟晶状体中，在两种上皮中 细胞和分化纤维。阻挡其他镜片的功能 连接蛋白，干扰基因的跨显性负结构 连接蛋白功能将在小鼠体内以转基因的形式表达，由 αA-晶状体蛋白启动子，靶向表达到晶状体。在 小鸡，活跃的跨显性负向构造将被引入 通过逆转录病毒感染发育的晶状体，使用复制- 强效RCAS-A禽流感病毒。在第二个特定目标中，磷酸化 连接蛋白上的位点将通过传统的肽图和 测序。确定关键的磷酸化丝氨酸和苏氨酸残基 通过这种作图将被突变并标记表位。这些连接蛋白 将突变株导入培养的晶状体细胞中，通过转基因和 使用RCAS逆转录病毒植入整个晶状体。变种人的能力 连接蛋白组装成通道，被输送到血浆 膜和靶向缝隙连接将紧随其后 在培养和发育中的晶状体中均有免疫组织化学染色。 将这些突变型连接蛋白导入通讯阴性细胞 Neuro2A神经母细胞瘤细胞株将允许研究单个细胞的变化 通道电导、电压门控和pH灵敏度 丢失特定的磷酸化残基。

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.