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是一个配体门控离子通道，介导神经肌肉接点（NMJ）的快速信号传递。这种受体蛋白在近20年前首次被纯化，但其原子结构仍不明确。在之前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，该系统允许以单体形式产生大量的x亚基胞外结构域（alpha211），以及以二聚体形式产生α和δ亚基的胞外结构域（α δ异二聚体）。生物化学和药理学研究表明，重组蛋白折叠成天然的构象，对胆碱能配体有很高的亲和力。此外，我们还优化了o211的核磁共振（NMR）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）配合的α δ异二聚体的小晶体的产生。作为我们过去研究的逻辑延伸，在这个竞争更新应用中提出的具体研究目标是：(1)通过多维核磁共振确定o211的高分辨率结构；(2)通过x射线衍射测定o2i异源二聚体与ACh或自身免疫抗体mAb35配合物的原子结构。阐明AChR的三维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的研究，包括GABA、甘氨酸和5-HT3受体。由于这些蛋白在疼痛、痴呆、癫痫和中风的发病机制中起作用，确定它们的结构对于合理设计更有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蛋白的结构，这是一种由运动神经元分泌的蛋白质，可诱导肌细胞膜上的achr聚集在NMJ处。多种形式的农用蛋白结合特性和生物活性不同，是通过多种组织中农用蛋白mrna的选择性剪接产生的。选择性剪接如何调节achr聚类活性是完全未知的。在这里，我们建议用核磁共振来解决神经和肌肉结构的c端结构域的溶液结构。这些结构信息将有助于恢复agfin在NMJ突触发生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.