The Neuronal Nicotinic Acetylcholine Receptor Interactome via a Knock-In Mouse

描述（由申请人提供）：本提案描述了一种创新的研究方法，旨在最终揭示产生和维持尼古丁依赖的分子机制。为了实现这一目标，我们建议通过结合新兴转基因和蛋白质组学技术的前沿特征，开发新的和可推广的研究工具，特别是通过在现有的新型敲入小鼠中使用新型药理学标记的含有α -3亚基的尼古丁受体来利用现代质谱的强大和复杂能力。这个项目是我们实验室的一个新项目，旨在将我们之前从结构和功能工作中获得的基本见解应用于药物成瘾领域的突出问题。对尼古丁诱导的细胞内蛋白质-蛋白质相互作用（即相互作用组）的改变的详细了解，涉及神经元尼古丁乙酰胆碱受体（nAChRs）的假设调节细胞质区域，有望为尼古丁强化、耐受性和致敏性的行为表现如何发展提供独特的有价值的见解。核心概念是，新型药理学标签敲入小鼠可以提供通用工具来研究与异常大的细胞质环相互作用的调节蛋白，这是所有nAChR亚型共同的特征。我们的alpha3敲入将α -班加罗毒素（Bgtx）结合位点引入到通常对Bgtx不敏感的神经元nachr中。结合Bgtx的能力将用于在温和条件下生物化学分离受体，以及相关的调节信号复合物。这些相关蛋白将使用串联质谱法鉴定，并绘制其磷酸化位点的功能图。为了严格测试这种方法，我们将重点关注在上颈神经节神经元原代培养中表达的bgtx敏感α亚基，其突触后含有α 3的nAChRs对突触传递至关重要。该系统将使我们能够探索尼古丁暴露对nAChR相互作用组谱的影响，并将为中枢神经系统中alpha3信号复合物的研究铺平道路；特别是在腹侧被盖区和内侧束，与尼古丁成瘾密切相关的区域。公共卫生相关性：了解尼古丁调节的细胞过程级联和蛋白质相互作用将有助于开发新的健康干预措施。

DESCRIPTION (provided by applicant): This proposal describes an innovative research approach aimed at ultimately revealing the molecular mechanisms that produce and maintain nicotine dependence. To accomplish this, we propose to develop new and generalizable investigative tools by combining cutting-edge features in emerging transgenic and proteomic technologies, specifically by using novel pharmatope-tagged alpha-3-subunit-containing nicotinic receptors in an existing novel knock-in mouse to harness the powerful and sophisticated capabilities of modern mass spectrometry . This project is a new venture in our lab that seeks to apply the fundamental insights gained from our previous structural and functional work to outstanding problems in the drug addiction area. A detailed understanding of nicotine-induced alterations in intracellular protein-protein interactions (i.e., the interactome) involving the putative regulatory cytoplasmic domain of neuronal nicotinic acetylcholine receptors (nAChRs) promises to provide uniquely valuable insights into how behavioral manifestations of nicotine reinforcement, tolerance and sensitization develop. The core concept is that novel pharmatope-tagged knock-in mice can provide general tools to study regulatory proteins interacting with the unusually large cytoplasmic loop that is a feature common to all nAChR subtypes. Our alpha3-knock-in introduces an alpha-bungarotoxin (Bgtx) binding site into neuronal nAChRs that are normally Bgtx- insensitive. The ability to bind Bgtx will be used to biochemically isolate the receptor under mild conditions, together with associated regulatory signaling complexes. These associated proteins will be identified using tandem mass spectrometry and their phosphorylation sites functionally mapped. To critically test this approach, we will focus on the Bgtx-sensitive alphas subunit expressed in primary cultures of superior cervical ganglionic neurons whose post-synaptic alpha3-containing nAChRs are essential for synaptic transmission. This system will allow us to probe the effects of nicotine exposure on the nAChR interactome profile, and will pave the way for studies of alpha3 signaling complexes in the CNS; in particular, in the ventral tegmental area and medial habenula, regions strongly implicated in nicotine addiction. Public Health Relatedness: Knowledge of the cascade of cellular processes and protein interactions regulated by nicotine will contribute to the development of new health interventions.