Structural studies of heteromeric nicotinic acetylcholine receptors

摘要 神经元烟碱乙酰胆碱受体是成瘾、心理健康和精神疾病的重要治疗靶点 和神经退行性疾病。这些五聚体配体门控离子通道是 Cys-环受体超家族，介导整个中枢和中枢神经系统的快速神经传递， 周围神经系统在这里，我们建议确定两个代表性的高分辨率结构， 神经元烟碱受体亚型烟碱受体的结构分析一直受到 真核生物膜蛋白重组表达的挑战。这些蛋白质通常表达在 含量低，纯化后不稳定。此外，大多数烟碱受体是专性异聚体 在许多情况下，这些异聚体可以组装成具有不同亚基比例的五聚体。这 混合化学计量的复杂性存在于所有Cys环受体家族中，但最好表征为 尼古丁受体结构的异质性导致生理上重要的，微调的 药理学和通道特性。在目标1中，我们建议开发表达和 这些受体可以以多种化学计量组装。在目标2和3中，我们建议： 应用这些方法来确定生理学上两种异聚烟碱受体的结构， 相关的和功能上不同的替代化学计量。单个受体结构将提供关键的 深入了解离子渗透和配体识别的结构基础。比较 两种受体的结构，以及相同受体的替代化学计量，将提供可靠的 结构基础，以了解各自独特的生物物理和药理学特性 受体亚单位组合。

Abstract Neuronal nicotinic acetylcholine receptors are essential therapeutic targets for addiction, mental health and neurodegenerative disorders. These pentameric ligand-gated ion channels are the prototypical members of the Cys-loop receptor superfamily, which mediate fast neurotransmission throughout the central and peripheral nervous systems. Here we propose to determine high-resolution structures of two representative neuronal nicotinic receptor subtypes. Structural analysis of nicotinic receptors has been hampered by the challenges of recombinant expression of eukaryotic membrane proteins. These proteins typically express at low levels and are unstable after purification. Furthermore, most nicotinic receptors are obligate heteromers and in many cases these heteromers can assemble as pentamers with different ratios of subunits. This complication of mixed stoichiometry is present among all Cys-loop receptor families but is best characterized in the nicotinic receptors. The structural heterogeneity results in physiologically important, finely tuned pharmacological and channel properties. In Aim 1, we propose to develop methods for expression and purification of these receptors that can assemble in multiple stoichiometries. In Aims 2 and 3 we propose to apply these approaches to determine structures of two heteromeric nicotinic receptors in physiologically- relevant and functionally-distinct alternate stoichiometries. The individual receptor structures will provide key insights into the structural underpinnings of ion permeation and ligand recognition. Comparison of the structures of the two receptors, and of alternative stoichiometries of the same receptor, will provide a reliable structural foundation for understanding the distinctive biophysical and pharmacological properties of each receptor subunit combination.