Expression and Structure of the Acetylcholine Receptor

描述（由申请人提供）：我们研究的长期目标是阐明烟碱乙酰胆碱受体（AChR）功能的分子机制。肌肉AChR是一种配体门控离子通道，介导神经肌肉接头（NMJ）的快速信号传递。这种受体蛋白在近20年前首次被纯化，但其原子结构仍然不清楚。在我们先前的NIH资助的支持下，我们已经确定了小鼠肌肉AChR α和δ亚基上的最小配体结合域。此外，我们已经建立了一个酵母表达系统，允许生产大量的细胞外结构域的x亚基的单体形式（α 211），以及细胞外结构域的α和δ亚基的二聚体形式（α adelta异源二聚体）。生物化学和药理学研究表明，重组蛋白折叠天然样构象与胆碱能配体的高亲和力。此外，我们已经优化了条件的均匀同位素标记和结构测定的o211的核磁共振（NMR）。结晶条件的初步筛选已导致与乙酰胆碱（ACh）复合的小晶体的产生。作为我们过去研究的逻辑延伸，在这一竞争性更新申请中提出的研究的具体目标是：（1）通过多维NMR确定o211的高分辨率结构;（2）通过X射线衍射解决与ACh或自身免疫抗体mAb 35复合的o2 i异源二聚体的原子结构。阐明AChR的3D结构是理解受体如何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，包括GABA，甘氨酸和5-HT 3受体的研究。由于这些蛋白质在疼痛、痴呆、癫痫和中风的发病机制中起作用，因此确定它们的结构对于合理设计更具选择性的治疗剂至关重要。我们研究的第三个具体目标是研究神经聚集蛋白的结构，神经聚集蛋白是由运动神经元分泌的一种蛋白质，它诱导乙酰胆碱受体在NMJ的肌细胞膜上聚集。在多种组织中，通过聚集蛋白mRNA的选择性剪接产生结合特性和生物活性不同的多种形式的聚集蛋白。选择性剪接如何调节乙酰胆碱受体聚集活性是完全未知的。在这里，我们建议解决的C-末端结构域的神经和肌肉聚集蛋白的NMR。这些结构信息将有助于恢复在NMJ突触发生过程中agfin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.