STRUCTURE AND FUNCTION OF LENS COMMUNICATING CHANNELS

镜头通信通道的结构和功能

• 批准号: 3258584
• 负责人: GUIDO A ZAMPIGHI
• 金额: $ 11.37万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-08-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3258584
• 关键词: axon; biological information processing; calmodulin; electron microscopy; gap junctions; image processing; immunocytochemistry; laboratory rat; lens; lens proteins; liver cells; membrane permeability; membrane reconstitution /synthesis

The ultimate purpose of the research proposed in this application is to understand the structural organization and mechanisms of action of gap junctions. These organelles are thought to mediate and regulate the passage of low molecular weight molecules and ions between adjacent cells in an enormous variety of animal tissues. The unraveling of the mechanisms involved in cell-cell communication will be fundamental to further our understanding of normal tissue physiology (i.e., cardiac muscle), development and cell differentiation, and pathological processes such as cancer and lens cataracts. Membranes isolated from lenses of calves and mature bovines contain junctions of different morphology (16-18nm thick, 12-14nm thick, and wavy) and protein composition. They are being studied by electron microscopy, immunocytochemistry, and biochemical and electrical methodology. Specifically, the studies proposed will focus on the localization and electrical characteristics of two putative junction-forming proteins of the lens: MIP26 (the main intrinsic protein of the lens) and MP70 (present in the cortex of lenses from mature animals and in both cortex and nucleus of lenses of calves). The electrical properties of MIP26 and MP70 can be studied independently since these proteins can be isolated separately by using non-ionic detergents at low concentrations. The proteins are then completely solubilized with high concentration of the non-ionic detergent octylglucoside, reconstituted into single-walled liposomes, and fused with planar lipid films. The single and multi-channel properties of these proteins are characterized and compared with each other and those of liver gap junction proteins reconstituted by others. This application also proposes to study the molecular architecture of the gap junction channel at the secondary structural level. Hydrophobicity plots of the primary structure of liver hepatocytes and heart myocite gap junction protein strongly suggest that each monomer is constructed of four intramembrane spanning domains. Similar plots calculated from the primary structure of lens MIP26 suggest six membrane spanning domains. Cryo-electron microscopy and computer image processing will be utilized to experimentally test these predictions.

本申请提出的研究的最终目的 就是要了解事物的结构、组织和机制 间隙连接的作用。 这些细胞器被认为可以介导 并调节低分子量分子的通过 多种动物中相邻细胞之间的离子 组织。 揭示细胞间的机制 沟通对于加深我们的理解至关重要 正常组织生理学（即心肌）、发育和 细胞分化和病理过程，例如癌症 和晶状体白内障。 从小牛和成熟牛的晶状体中分离出的膜 包含不同形态的结（16-18nm 厚，12-14nm 厚且呈波浪状）和蛋白质成分。 他们正在被研究 通过电子显微镜、免疫细胞化学和生化 电气方法论。 具体来说，拟议的研究将 重点关注两种产品的定位和电气特性 晶状体的假定连接形成蛋白：MIP26（主要 晶状体的内在蛋白质）和 MP70（存在于成熟动物的晶状体皮质中， 小腿晶状体的皮质和核）。 电气 MIP26 和 MP70 的特性可以独立研究，因为 这些蛋白质可以通过使用非离子分离器单独分离 低浓度的洗涤剂。 那么蛋白质是 高浓度非离子完全溶解 洗涤剂辛基葡萄糖苷，重构为单壁 脂质体，并与平面脂质膜融合。 单身和 这些蛋白质的多通道特性被表征和 相互比较以及肝脏间隙连接蛋白的比较 被别人重组。 该申请还建议研究分子 次级间隙连接通道的结构 结构层面。 一级结构的疏水性图 肝细胞和心肌间隙连接蛋白的 强烈建议每个单体由四个单体构成 膜内跨域。 类似的图计算自 晶状体 MIP26 的主要结构表明有六个跨膜 域。 冷冻电子显微镜和计算机图像处理 将用于通过实验来测试这些预测。