LENS METABOLIC COOPERATION GAP JUNCTIONS AND CATARACT

晶状体代谢协作间隙连接和白内障

• 批准号: 2378035
• 负责人: DANIEL A. GOODENOUGH
• 金额: $ 35.26万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2378035
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus; alternatives to animals in research; biological signal transduction; cataract; cell cell interaction; chick embryo; electrophysiology; embryo /fetus tissue /cell culture; gap junctions; gene expression; gene targeting; homeostasis; immunocytochemistry; laboratory mouse; lens proteins; membrane proteins; nucleic acid sequence; polymerase chain reaction; protein sequence; protein structure function; retinal pigment epithelium; transfection

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.

由于其独特的结构和功能，透镜的代谢和离子 体内平衡完全依赖于保持相互连接的透镜纤维 通过缝隙连接通讯途径到达表面上皮细胞。 为了避免白内障，离子稳态至关重要：沉淀 透镜纤维中高浓度的可溶性蛋白质 细胞增殖 这项建议的长期目标是 通过实验证明细胞间通讯的功能， 透镜发育和体内平衡中的缝隙连接。 实验 它们被设计成特定的连接蛋白块， 在透镜中起作用。 Cx43基因敲除小鼠现已上市 在商业上。 这些动物在出生时死于心脏缺陷， 足够长的时间来研究 首先在正常透镜中表达的Cx43敲除的后遗症 发育，并坚持在成熟的透镜，在上皮 细胞和分化纤维。 阻挡其他透镜的功能 连接蛋白，反式显性负结构，干扰 连接蛋白功能将在小鼠中作为转基因表达， α A-晶状体蛋白启动子，以靶向表达到透镜。 在 鸡，将活性反式显性阴性构建体引入 通过逆转录病毒感染发育中的透镜，使用复制- RCAS-A禽流感病毒。 在第二个具体目标中，磷酸化 连接蛋白上的位点将通过常规的肽作图进行作图， 测序 鉴定的关键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 通过转染将突变体引入培养的透镜细胞中， 植入到整个晶状体中 变种人的能力 连接蛋白组装成通道， 膜和目标间隙连接将遵循 免疫组织化学在这两个文化和发展中的镜头。 将这些突变的连接蛋白转染到通讯阴性的 Neuro 2A神经母细胞瘤细胞系将允许研究单个神经母细胞瘤的变化。 通道电导、电压门控和pH敏感性， 特定磷酸化残基的丢失。