MECHANISMS OF ACETYLCHOLINE RECEPTOR LOCALIZATION

乙酰胆碱受体定位机制

• 批准号: 3396096
• 负责人: ROBERT W SEALOCK
• 金额: $ 20.41万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3396096
• 关键词: Torpedo; acetylcholine; alternatives to animals in research; basolateral membrane; cell cell interaction; cell membrane; cytoskeletal proteins; dystrophin; freeze etching; histochemistry /cytochemistry; immunocytochemistry; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; monoclonal antibody; muscle proteins; muscular dystrophy; neuromuscular junction; nicotinic receptors; protein structure; sarcolemma; striated muscles

DESCRIPTION (Investigator's abstract): The location, organization and lateral mobility of nicotinic acetylcholine receptors at the neuromuscular junction are believed to be controlled by a transmembrane complex of cytoskeletal and basal lamina elements. Thus far, only a few proteins which presumably form part of this complex have been identified. To advance the hypothesis further, we need to identify more of these proteins and to see how they interact together. This project aims at these questions through identification and characterization of proteins in the corresponding complex in electric tissue of electric rays, a muscle-derived tissue. In one part, the project focuses on 2 proteins already identified, namely, a 58kD protein and the electric tissue homolog of dystrophin, the protein of normal muscle which is absent or defective in Duchenne and Becker muscular dystrophy. Whether these proteins occur in a complex with actin and other proteins on the postsynaptic membrane will be determined. In the second part, new proteins will be identified by monoclonal antibody methods. The immunogen will be material extracted from a more intact preparation of electric tissue postsynaptic membranes than has been used heretofore. The antibodies so generated, as well as antibodies already available against dystrophin and the 58kD protein, will be used to localize identified proteins relative to accumulations of receptor and to membrane-associated structural elements in electric tissue and muscle. The methods will be immunofluorescence and immunoelectron microscopy (thin section and freeze-etch methods). The identified proteins will be purified by immunoaffinity methods for determination of their molecular morphologies by electron microscopy and for identification of copurifying proteins with which they may interact. In this way, we hope to identify cytoplasmic and transmembrane components of the transmembrane complex and their shapes. Equally important, we can expect to identify additional proteins which, like the 58kD protein and dystrophin, are components of the non-synaptic sarcolemma as well as the neuromuscular junction. Any of these proteins could be potentially involved in the action of dystrophin to protect the sarcolemma against mechanical damage during contraction. This action is also probably mediated through a transmembrane complex between dystrophin and extracellular materials. The proposed project thus may have direct bearing on two problems at once, one of these being relevant to a major disease.

描述（调查员的摘要）：位置，组织和 烟碱乙酰胆碱受体在神经肌肉的横向迁移率 据信连接受到跨膜复合物的控制 细胞骨架和基底层薄片元素。 到目前为止，只有少数蛋白质 大概已经确定了这一复合物的一部分。 到 进一步推进了假设，我们需要确定更多这些蛋白质 并了解它们如何一起互动。 这个项目针对这些 通过识别和表征蛋白质中的问题 电射线电动组织中的相应复合物，肌肉衍生 组织。 在一部分中，该项目重点是已经确定的2种蛋白质， 即，一种58KD蛋白和肌营养不良蛋白的电组织同源物， 正常肌肉的蛋白质，在杜钦（Duchenne）中没有或有缺陷 贝克尔肌肉营养不良。 这些蛋白是否发生在一个复合物中 将确定突触后膜上的肌动蛋白和其他蛋白质。 在第二部分中，新蛋白将通过单克隆抗体鉴定 方法。 免疫原将是从更完整的材料中提取的 与已使用的电动组织的准备相比 迄今为止。 如此生成的抗体以及抗体 可用于肌营养不良蛋白和58KD蛋白，可用于本地化 相对于受体的积累，已鉴定的蛋白质和 电组织和肌肉中与膜相关的结构元素。 这 方法将是免疫荧光和免疫电子显微镜（薄） 部分和冻结方法）。 确定的蛋白质将被纯化 通过确定其分子形态的免疫亲和力方法 通过电子显微镜和用于鉴定与 他们可能会互动。 这样，我们希望识别细胞质和 跨膜复合物及其形状的跨膜成分。 同样重要的是，我们可以期望鉴定出其他蛋白质，这些蛋白质， 像58KD蛋白和肌营养不良蛋白一样，是非突触的成分 肌膜和神经肌肉结。 这些蛋白质中的任何一个 可能参与肌营养素保护的作用 收缩期间针对机械损伤的肌膜。 这个动作是 也可能通过肌营养不良蛋白之间的跨膜复合物介导 和细胞外材料。 因此，拟议的项目可能有直接 同时解决两个问题，其中一个与专业有关 疾病。