Developing nanobody immune libraries against native neuronal nicotinic receptor complexes

摘要 烟碱型乙酰胆碱受体在大脑中无处不在，是调节注意力和觉醒的基础，通过 胆碱能神经元。NAChRs在强化和奖赏通路的神经元中高度表达，并且 药物暴露对nAChRs的适应不良影响导致药物成瘾。这些受体是 对尼古丁(一种nAChRs的直接正构体激动剂)和其他滥用药物的成瘾至关重要 由药物引起的奖赏中心胆碱能活动的可塑性变化所致。持之以恒的挑战 面对任何以nAChR为靶向的药物治疗成瘾的方法是在组装和 大脑表达形式的蛋白质的分布。这个项目的长期目标是开发一种 准确而全面的方法学来识别和治疗原生nAChR的整个家族 构象。这个R03提案的短期目标是证明酵母展示的纳米体 文库，针对两种不同的、广泛表达的和生理上关键的nAChRs构象进行筛选， 将展示在体外荧光中强有力地解析nAChR的两个单独的化学计量比的能力 以及电生理学检测。其基本原理是提供概念证明，该方法可以具体 针对单个受体类型，以刺激成功鉴定其受体类型的试剂的进一步表征 修改nAChR活动的可能功能，并最终将该方法确立为可行的 方法研究天然nAChRs整个家族的天然表达和组装特性。这 这项研究包括两个相关的目标：1)开发富含酵母安装的可变区的纳米体库 有效结合纯化的42 nAChRs和2)以建立有效的反筛选的结构域模块 去除化学计量学上的非特异性纳米小体以纯化选择性靶向两个 生理上相关的、结构上不同的42组件。这代表了 先进的酵母菌纳米体筛选方法克服了理解 烟碱受体蛋白表达和分布的不适应性变化。开发和交付 作为这项研究的总体目标，在体内研究和操纵所有天然nAChR的生物工具箱将 代表着一项重大的生物医学进步，用于研究和抗击成瘾和其他人类疾病 与胆碱能神经系统有关。

ABSTRACT Nicotinic acetylcholine receptors are ubiquitous in the brain and underlie regulation of attention and arousal by cholinergic neurons. nAChRs are highly expressed in neurons of the reinforcement and reward pathways, and maladaptive effects on nAChRs resulting from drug exposure contribute to drug addiction. These receptors are crucial in addiction both to nicotine (a direct orthosteric agonist of nAChRs) and to other drugs of abuse resulting from drug induced plastic changes in cholinergic activity in reward centers. A persistent challenge facing any nAChR-targeted pharmaceutical approach against addiction is the broad diversity in assembly and distribution of the brain-expressed forms of the protein. The long-term goal of this project is to develop an accurate and comprehensive methodology to identify and therapeutically target the full family of native nAChR conformations. The short-term goal of this R03 proposal is to demonstrate that a yeast-display nanobody library, screened against two distinct, widely expressed and physiologically critical conformations of nAChRs, will demonstrate the ability to robustly parse two separate stoichiometries of the nAChR in in vitro fluorescence and electrophysiology assays. The rationale is to deliver proof-of-concept that this approach can specifically target individual receptor types, to stimulate further characterization of successfully identified reagents for their possible functionality in modifying nAChR activity, and ultimately to establish the approach as a feasible method to study the native expression and assembly properties of the entire family of native nAChRs. This study includes two related objectives: 1) to develop a nanobody library enriched for yeast-mounted variable domain modules that potently bind purified 42 nAChRs and 2) to establish an effective counter-screening assay to deplete stoichiometrically non-specific nanobodies to purify interactors that selectively target two physiologically relevant, structurally distinct assemblies of 42. This represents an innovative application of the cutting-edge yeast nanobody screening method against a persistent roadblock in understanding the maladaptive changes to nicotinic receptor protein expression and distribution. Development and delivery of a biologic toolbox to study and manipulate all native nAChRs in vivo, as is the overall goal of this research, would represent a significant biomedical advance to investigate and combat addiction and other human ailments associated with the cholinergic nervous system.