STRUCTURE/FUNCTION ANALYSIS OF ACCHR EPITOPES

该计划项目的中心主题是使用单克隆 针对确定的寡肽的抗体(MAbs) 烟碱型乙酰胆碱受体(AcChR)亚基AS AcChR结构的位点特异性探针(初级和 三级结构)和AcChR功能。我们将特别注意 支付给其绑定干扰此功能的单抗 跨膜糖蛋白神经递质受体。项目 1-4将评估单抗对以下特定方面的影响 AcChR功能：项目1-配体结合，项目2-离子通量 停流技术；项目3-离子通道功能由 膜片钳技术；项目4-神经肌肉传导 体外和体内。项目5和6将使以下表位本地化 AcChR结构中的这些单抗：项目5-生产 合理选择模拟特定基因的人工合成寡肽 AcChR亚单位内的站点，评估 单抗表位的糖基部分，项目6-鉴定 结合mAbs的低分辨率三维模型 小角X射线衍射法和高分辨率AcChR法 电子显微镜，以及荧光探针的定位。 科学核心将产生新的针对 在项目5生产的多肽，将提供纯化的， 针对不同项目的特色化单抗。在项目4中， 多肽可能被用于开发一种新的治疗方法 实验性重症肌无力的模式。本计划 该项目代表了免疫学家的合作努力， 生物化学家、电生理学家、多肽化学家和 物理化学家/结构化学家的信息来自 各种项目放在一起，将允许绘制各种不同的地图 AcChR初级序列中的位点，特别是在功能上 重要地点，到三维模型。

The central theme of this Program Project is to use monoclonal antibodies (mAbs) directed against defined oligopeptides within the subunits of the nicotinic acetylcholine receptor (AcChR) as site-specific probes of AcChR structure (both primary and tertiary structure) and AcChR function. Special attention will be paid to mAbs whose binding interferes with the function of this transmembrane glycoprotein neurotransmitter receptor. Projects 1-4 will assess the effects of the mAbs on particular aspects of AcChR function: Project 1 - ligand binding, Project 2 - ion fluxes by stopped flow technique; Project 3 - ion channel function by patch clamp technique; Project 4 - neuromuscular transmission in vitro and in vivo. Projects 5 and 6 will localize the epitopes of these mAbs within the AcChR structure: Project 5 - production of rationally chosen synthetic oligopeptides mimicking specific sites within AcChR subunits, assessment of the contribution of glycosyl moieties to mAb epitopes, Project 6 -identification of bound mAbs on the low resolution three-dimensional model of AcChR by both small angle X-ray diffraction and high resolution electron microscopy, as well as localization of fluorescent probes. The Scientific Core will produce new mAbs directed against the peptides produced in Project 5 and will provide purified, characterized mAbs for the various projects. In Project 4, the peptides will be used to possibly develop a new treatment modality for experimental myasthenia gravis. This Program Project represents a collaborative effort of immunologists, biochemists, electrophysiologists, peptide chemists, and physical/structural chemists in which the information from the various projects, taken together, will allow for mapping of various sites in the primary sequence of AcChR, especially functionally important sites, onto the three-dimensional model.