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

描述(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技术和蛋白质组技术的尖端特征来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的含α-3亚单位的烟碱受体来利用现代质谱学的强大和复杂的能力。这个项目是我们实验室的一个新项目，旨在将我们以前的结构和功能工作中获得的基本见解应用于吸毒成瘾领域的突出问题。详细了解尼古丁诱导的细胞内蛋白-蛋白相互作用(即相互作用组)的变化，涉及神经元烟碱型乙酰胆碱受体(NAChRs)的胞浆调节域，有望为尼古丁强化、耐受和敏化的行为表现提供独特的有价值的见解。其核心概念是，新型的药膜标记敲入小鼠可以提供通用的工具来研究与异常大的细胞质环相互作用的调控蛋白，这是所有nAChR亚型的共同特征。我们的α3敲入将α-银环蛇毒素(BGTX)结合位点引入到神经元nAChRs中，而神经元nAChRs通常对BGTX不敏感。结合BGTX的能力将用于在温和的条件下生化分离受体以及相关的调节信号复合体。这些相关蛋白质将使用串联质谱仪进行鉴定，并对它们的磷酸化位点进行功能定位。为了严格测试这一方法，我们将关注在原代培养的颈上神经节神经元中表达的对BGTX敏感的α亚基，其突触后含有α3的nAChRs对于突触传递是必不可少的。该系统将使我们能够探索尼古丁暴露对nAChR相互作用组的影响，并将为研究中枢神经系统中的Alpha3信号复合体铺平道路；特别是在腹侧被盖区和内侧缰核，这两个区域与尼古丁成瘾密切相关。公共卫生相关性：尼古丁调节的细胞过程和蛋白质相互作用的级联知识将有助于开发新的卫生干预措施。

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.