MOLECULAR MECHANISM OF ACETYLCHOLINE RECEPTOR CLUSTERING

本提案的总体目标是阐明 神经诱导乙酰胆碱受体的分子机制 聚类 神经诱导的AChR聚集是其中的关键步骤之一 参与神经肌肉接头（NMJ）的形成。 我们 建议研究AChR聚类的复杂过程， 简化了神经和肌肉细胞。 神经细胞会 被假定的聚类代理所取代。这些将是 神经组织的衍生物或来自肌肉细胞的基底层，或 已被证明具有AChR聚集的鱼雷电器官 在培养的肌肉细胞中的活性。 肌肉细胞会 被培养的小鼠成纤维细胞所取代， 功能性细胞表面AChR（AChR-成纤维细胞）。 该系统具有 许多优于标准神经-肌肉共培养系统的优点 用于研究乙酰胆碱受体聚集。 没有神经或肌肉细胞 成熟变化，2）无神经或基底膜因素其他 3）没有内在肌细胞蛋白 除了乙酰胆碱受体和那些专门为 study. 这项建议的具体目标可分为 三大项目。 项目#1将检测推定的AChR 聚类剂的能力，以诱导乙酰胆碱受体簇上的 AChR-成纤维细胞的表面。 AChR成纤维细胞将被孵育 与集群代理和集群形成将是 使用荧光标记定量评价， 显微镜和计算机分析。 #20002;定，决定 如果集群代理通过直接交互诱导集群 与AChR或通过与靶标的非AChR组分相互作用 细胞膜或细胞骨架。 成纤维细胞将暴露于 群集代理，然后在 诱导成纤维细胞表达表面AChR。 如果集群 不形成，这将是暗示，聚类代理直接 与乙酰胆碱受体相互作用。 如果星系团真的形成了， AChR不是群集剂的主要靶标。 项目#3将确定哪些AChR亚基和结构域， 亚基参与聚类。 我们会培养成纤维细胞 表达特异性突变AChRs的细胞系， 那些功能齐全，但失去了能力， 聚类;聚类中的任何缺陷都可以归因于突变的 亚基或结构域。 乙酰胆碱受体聚集及其在神经肌肉中作用的研究 突触发生具有广泛的医学意义。 的能力 理解和操纵NMJ的AChR动力学可能是有用的 在预防和治疗肌萎缩侧索硬化症等疾病中， 硬化症、炎性肌病和肌营养不良症， 神经肌肉传递衰竭引起的呼吸衰竭. 横膈膜上的NMJ 此外，NMJ及其AChRs是一种 神经-神经突触和配体门控受体模型 参与神经突触传递的通道。 因此，在本发明中， 研究运动神经元发挥其作用的机制， 对肌肉细胞的影响可能有助于阐明机制 中枢神经系统的神经元通过这种方式对每个神经元施加影响， 另一种是通过突触。 这种影响可能对 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.