Structural studies of heteromeric nicotinic acetylcholine receptors

摘要 神经元烟碱型乙酰胆碱受体是成瘾、心理健康的重要治疗靶点 和神经退行性疾病。这些五聚体配基门控离子通道是典型的成员 是Cys-loop受体超家族的一员，它在中枢和中枢神经系统中介导快速的神经传递。 周围神经系统。在这里，我们建议确定两个具有代表性的高分辨率结构 神经元烟碱受体亚型。尼古丁受体的结构分析一直受到 真核膜蛋白重组表达面临的挑战。这些蛋白质通常在 含量低，纯化后不稳定。此外，大多数烟碱受体都是专性异构体。 在许多情况下，这些异构体可以组装成具有不同亚基比例的五聚体。这 混合化学计量学的并发症在所有Cys-loop受体家族中都存在，但最好的特征是 尼古丁受体。结构的异质性导致了生理上的重要，微调 药理和通道特性。在目标1中，我们建议开发表达和 纯化这些可以在多种化学计量比中组装的受体。在目标2和目标3中，我们建议 应用这些方法确定生理上的两种异构型烟碱受体的结构 相关的和功能不同的替代化学计量学。单个受体结构将提供关键 对离子渗透和配体识别的结构基础的洞察。两个版本的比较 两种受体的结构，以及同一受体的不同化学计量比，将提供可靠的 了解每种化合物独特的生物物理和药理性质的结构基础 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.