Expression and Structure of the Acetylcholine Receptor

描述(申请人提供)：我们研究的长期目标是阐明烟碱型乙酰胆碱受体(AChR)功能的分子机制。肌肉AChR是一种配体门控离子通道，在神经肌肉接头(NMJ)介导快速信号传递。近20年前，该受体蛋白首次被提纯，但其原子结构仍不明确。在我们先前NIH资助的支持下，我们已经确定了小鼠肌肉AChR的α和Delta亚基上的最小配体结合域。此外，我们建立了一个酵母表达系统，允许以单体形式(α211)生产大量x亚基的胞外域，以二聚体形式生产α和德尔塔亚基的胞外域(α-增量异二聚体)。生化和药理学研究表明，重组蛋白以类天然构象折叠，与胆碱能配体有很高的亲和力。此外，我们还优化了用核磁共振方法对o211进行均匀同位素标记和结构测定的条件。结晶条件的初步筛选导致了与乙酰胆碱(ACh)形成络合物的α-三角洲杂二聚体的小晶体的产生。作为我们过去研究的逻辑延伸，在这一竞争性更新应用中提出的具体研究目标是：(1)用多维核磁共振确定O211的高分辨结构；(2)用X射线衍射法解决O2I与ACh或自身免疫抗体mAb35形成的络合物中的原子结构。阐明AChR的三维结构是了解受体与配体相互作用的关键。这些信息也可能适用于其他配体门控离子通道的研究，包括GABA、甘氨酸和5-HT3受体。由于这些蛋白质在疼痛、痴呆、癫痫和中风的发病机制中发挥作用，因此确定它们的结构对于合理设计更具选择性的治疗药物至关重要。我们研究的第三个具体目的是研究神经集聚蛋白的结构，这是一种由运动神经元分泌的蛋白质，可以诱导AChRs在NMJ的肌肉细胞膜上聚集。通过在各种组织中选择性地剪接集聚蛋白mRNAs，可以产生多种形式的集聚蛋白，它们的结合性质和生物活性不同。选择性剪接如何调节AChR聚集活性是完全未知的。在这里，我们建议用核磁共振来解决神经和肌肉agrins的C末端结构域的溶液结构。这些结构信息将有助于恢复agfin在NMJ突触形成过程中重要功能的分子机制。

DESCRIPTION (provided by applicant): The long-term objective of our research is to elucidate the molecular mechanisms underlying the function of nicotinic acetylcholine receptors (AChR). The muscle AChR is a ligand-gated ion channel that mediates fast signal transmission at the neuromuscular junction (NMJ). The receptor protein was first purified nearly two decades ago, but its atomic structure remains poorly defined. With the support of our previous NIH grant, we have identified the minimal ligand-binding domains on the alpha and delta subunits of mouse muscle AChR. In addition, we have established a yeast expression system that allows the production of large quantities of the extracellular domains of x subunit in monomeric form (alpha211) as well as the extracellular domains of both alpha and delta subunits in dimeric form (alphadelta heterodimer). Biochemical and pharmacological studies have demonstrated that the recombinant proteins fold in native-like conformation with high affinity to cholinergic ligands. Furthermore, we have optimized the conditions for uniform isotopic labeling and structural determination of o211 by nuclear magnetic resonance (NMR). Initial screening of conditions for crystallization has led to the production of small crystals of alphadelta heterodimers in complex with acetylcholine (ACh). As the logical extension of our past studies, the specific aims of research proposed in this competing renewal application are: (1) to determine the high-resolution structure of o211 by multidimensional NMR; and (2) to solve the atomic structure of o2i heterodimer in complex with ACh or the autoimmune antibody mAb35 by Xray diffraction. Elucidation of the 3D structure of AChR holds the key to understanding how the receptor interacts with ligands. Such information may also be applicable to studies of other ligand-gated ion channels including GABA, glycine and 5-HT3 receptors. As these proteins play a role in the pathogenesis of pain, dementia, epilepsy, and stroke determination of their structure is essential for the rational design of more selective therapeutic agents. The third specific aim of our research is to study the structure of neural agrin, a protein secreted by motoneurons that induces clustering of AChRs on muscle cell membrane at the NMJ. Multiple forms of agrin that differ in binding properties and bioactivity are generated through alternative splicing of agrin mRNAs in a variety of tissues. How alternative splicing regulates AChR-clustering activity is completely unknown. Here, we propose to solve the solution structure of the C-terminal domains of both neural and muscle agrins by NMR. The structural information will help to recover the molecular mechanisms underlying the important function of agfin during synaptogenesis at the NMJ.