MOLECULAR MECHANISM OF ACETYLCHOLINE RECEPTOR CLUSTERING

这项提案的总体目标是澄清 神经诱导型乙酰胆碱受体的分子机制 集群化。神经诱导的乙酰胆碱受体聚集是关键步骤之一 参与神经肌肉接头(NMJ)的形成。我们 建议通过大量的实验研究AChR聚簇的复杂过程 简化了神经和肌肉细胞。神经细胞会 取而代之的是可能的聚集剂。这些将是 神经组织或肌细胞基板的衍生物或 已被证明具有AChR聚集性的鱼雷电器官 培养的肌肉细胞的活性。肌肉细胞将是 被稳定表达的培养的小鼠成纤维细胞取代 功能性细胞表面AChRs(AChR-成纤维细胞)。这个系统有 与标准的神经-肌肉共培养系统相比，有许多优势 用于研究AChR聚集性。没有神经或肌肉细胞 成熟性改变，2)无神经或基底板因素 3)没有内在的肌肉细胞蛋白 除了AChR和专门为 学习。 这项建议的具体目的可以分为 三大工程。项目1将检测推定的AChR 聚集剂在体内诱导AChR簇的能力 AChR-成纤维细胞表面。Achr-成纤维细胞将被培养 使用聚集剂，形成的聚集体将是 使用荧光标记进行定量评估， 光学显微镜和计算机分析。项目2将决定 如果聚集剂通过直接相互作用诱导聚集 使用AChR或通过与目标的非AChR组件交互 细胞膜或细胞骨架。成纤维细胞将暴露在 然后，该代理将在 诱导成纤维细胞表达表面AChRs。如果集群出现这种情况 不形成，则会提示聚集剂直接 与AChRs相互作用。如果星系团确实形成了，它将表明 AChR不是群集代理的主要目标。 项目3将确定哪些AChR亚单位和结构域 亚基参与了聚类。我们将开发成纤维细胞 表达特异突变的AChRs的细胞系及其分析 它们功能齐全，但已经失去了 簇；簇中的任何缺陷都可以归因于突变的 亚单位或结构域。 乙酰胆碱受体聚集及其在神经肌肉中的作用研究 突触发生具有广泛的医学意义。一种能力 在NMJ上理解和操纵AChR动力学可能是有用的 预防和治疗肌萎缩侧索硬化症等疾病 硬化症、炎症性肌病和肌营养不良 由神经肌肉传递失败所致的呼吸衰竭 NMJ在横隔膜上。此外，NMJ及其AChR是一个 神经-神经突触模型与配体门控受体 参与神经-神经突触传递的通道。因此， 运动神经元发挥功能的机制研究 对肌肉细胞的影响可能有助于阐明其机制。 中枢神经系统神经元对每个神经元的影响 另一些则跨越突触。这样的影响可能对 学习和记忆的发展。

The overall goal of this proposal is to elucidate the molecular mechanism of nerve-induced acetylcholine receptor (AChR) clustering. Nerve-induced AChR clustering is one of the key steps involved in formation of the neuromuscular junction (NMJ). We propose to study the complex process of AChR clustering by greatly simplifying both the nerve and muscle cell. The nerve cell will be replaced by putative clustering agents. These will be derivatives of neural tissue or basal lamina from muscle cells or Torpedo electric organ that have been shown to have AChR clustering activity in cultured muscle cells. The muscle cell will be replaced by cultured mouse fibroblasts that stably express functional cell surface AChRs (AChR-fibroblasts). This system has a number of advantages over standard nerve-muscle coculture systems for studying AChR clustering. There are 1) no nerve or muscle cell maturational changes, 2) no nerve or basal lamina factors other than those introduced and 3) no intrinsic muscle cell proteins other than the AChR and those specifically introduced for the study. The specific aims of this proposal can be divided into three major projects. Project #1 will assay putative AChR clustering agents for their ability to induce AChR clusters on the surface of AChR-fibroblasts. AChR-fibroblasts will be incubated with clustering agents and the clusters formed will be quantitatively evaluated using fluorescent labelling, photomicroscopy and computer analysis. Project #2 will determine if the clustering agents induce clustering by direct interaction with AChR or by interaction with non-AChR components of the target cell membrane or cytoskeleton. Fibroblasts will be exposed to clustering agents and then the agent will be removed before the fibroblasts are induced to express surface AChRs. If clusters do not form, it will be suggestive that the clustering agent directly interacts with AChRs. If clusters do form, it will suggest that the AChR is not the primary target of the clustering agent. Project #3 will determine which AChR subunits and domains of subunits are involved in clustering. We will develop fibroblast cell lines that express specifically mutated AChRs and analyze those which are fully functional, but have lost the ability to cluster; any defects in clustering can be attributed to the mutated subunit or domain. Study of AChR clustering and its role in neuromuscular synaptogenesis has wide ranging medical significance. The ability to understand and manipulate AChR dynamics at the NMJ may be useful in preventing and treating such diseases as amyotrophic lateral sclerosis, inflammatory myopathies and muscular dystrophies and respiratory failure due to neuromuscular transmission failure at NMJs in the diaphragm. In addition, the NMJ and its AChRs are a model for nerve-nerve synapses and the ligand-gated receptor channels involved in nerve-nerve synaptic transmission. Thus, study of the mechanisms by which the motoneuron exerts its influence upon the muscle cell may help elucidate the mechanisms by which central nervous system neurons exert influences upon each other across synapses. Such effects may be important for the development of learning and memory.