EXPRESSION AND STRUCTURE OF THE ACETYLCHOLINE RECEPTOR

我们的长期目标是阐明 利用肌肉的配体-神经递质受体相互作用 烟碱乙酰胆碱受体（AChR）作为模型。尽管AChR是 第一次纯化和克隆近20年前，详细的结构 关于这种蛋白质的信息仍然非常有限。这主要是由于 使膜蛋白结晶的固有困难。因此，或主要 目的是表达大量的可溶性，N-末端 AChR α和δ亚基的胞外结构域，以及 确定它们的三维结构。这个领域，在阿尔法 亚基，包括几乎一半的亚基质量，并含有结合 α-银环蛇毒素（α-BuTx）以及所谓的主要 免疫原性区域（MIR）。这一区域是自身免疫的目标 重症肌无力中的抗体此外，此域还包含 与δ亚基组装形成α- δ异二聚体，其可以结合胆碱能配体，例如 乙酰胆碱（ACh）和d-筒箭毒碱（dTC）。具体目标是 建议是：1）确定α上的最小可溶性序列 可以折叠以形成α-BuT结合位点和MIR的亚基; 2） 定义α和结构域亚基上的最小结构域， 具有高亲和力乙酰胆碱结合位点的可溶性异源二聚体。妥善 折叠的α N-末端结构域和β-adelta异二聚体将是 在酵母中表达为分泌蛋白，并纯化至均一; 3） 确定α细胞外结构域的3D溶液结构， 游离形式和与α-BuTx的络合物。 此外，在Aidadelta异二聚体与 将使用转移的NOE描绘ACh和dTC; 4）使 A.A.A.异源二聚体-ACh复合物和MIR-自身免疫抗体复合物 以便使用X射线衍射方法研究它们的结构。 该项目的完成将使我们能够解决长期寻求的 AChR的配体结合位点的结构。它还将提供 深入了解决定功能的共同结构要素 其他配体门控离子通道，如GABA、甘氨酸和5-HT/3 受体。由于这些蛋白质在糖尿病的发病机制中起着重要作用， 疼痛，精神疾病和其他常见疾病，如癫痫和 脑卒中时，了解其结构对于合理用药至关重要 设计更有选择性的治疗药物。最后，技术要 通过这项工作开发的应有助于未来的结构研究， 这些和其他配体门控离子通道。

Our long-term goal is to elucidate the molecular mechanisms underlying ligand-neurotransmitter receptor interactions utilizing the muscle nicotinic acetylcholine receptor (AChR) as a model. Although the AChR was first purified and cloned nearly 20 years ago, detailed structural information of this protein is still very limited. This is mainly due to the inherent difficulty of crystallizing membrane proteins. Hence, or main objective is to express large quantities of soluble, N-terminal extracellular domains of the AChR alpha and delta subunits, and to determine their 3-dimensional structure. This domain, in the alpha subunit, comprises almost half the subunit mass and contains the binding site for alpha-bungarotoxin (alpha-BuTx) as well as the so-called major immunogenic region (MIR). This region is the target of autoimmune antibodies in myasthenia gravis. In addition, this domain contains the structural information for assembly with the delta subunit to form alpha- delta heterodimers which can bind cholinergic ligands, such as acetylcholine (ACh) and d-tubocurarine (dTC). The specific aims of this proposal are: 1) to identify a minimal soluble sequence on the alpha subunit that can fold to form an alpha-BuT binding site and the MIR; 2) to define the minimal domains on the alpha and domain subunits that can form a soluble heterodimer with a high affinity ACh-binding site. Properly folded alpha N-terminal domain and the alphadelta heterodimer will be expressed as secretory proteins in yeast, and purified to homogeneity; 3) to determine the 3D solution structure of the alpha extracellular domain in free form and in complex with alpha-BuTx using multi-dimensional NMR. Also, structural elements on the alphadelta heterodimer interaction with ACh and dTC will be delineated using transferred NOE; 4) to crystalize the alphadelta heterodimer-ACh complex and the MIR-autoimmune antibody complex in order to study their structure using x-ray diffraction methods. Completion of this project will allow us to solve the long-sought structure of the ligand-binding sites of the AChR. It will also provide insight into the common structural elements that determine the function of other ligand-gated ion channels, such as the GABA, glycine and 5-HT/3 receptors. As these proteins play important roles in the pathogenesis of pain, mental illness and other common disorders such as epilepsy and stroke, information on their structure is essential for the rational drug design for more selective therapeutic agents. Finally, techniques to be developed through this work should facilitate future structural studies of these and other ligand-gated ion channels.