Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.

α7烟碱型乙酰胆碱受体（α7-nicotinic acetylcholine receptor, α7-nAChR）是一类在脊椎动物中广泛表达的配体门控离子通道，同时也是哺乳动物中枢神经系统内主要的高亲和力α-银环蛇毒素（α-bungarotoxin, α-bgtx）结合蛋白。α7-nAChR可与多种调控其功能特性的蛋白发生相互作用。α7-nAChR相互作用组指的是在蛋白质复合物中与α7-nAChR存在相互作用或结合关系的全部蛋白的总和。为鉴定神经组织中的α7-nAChR相互作用组，本研究采用α-银环蛇毒素亲和磁珠，从野生型及α7-nAChR基因敲除（α7 KO）小鼠的全脑组织匀浆中分离得到α-银环蛇毒素亲和蛋白复合物。将亲和沉淀得到的蛋白经胰蛋白酶酶解后，利用Orbitrap Fusion质谱仪进行分析。通过α7-nAChR特异性配体α-银环蛇毒素分离得到的蛋白，被认定为α7-nAChR相关蛋白。本研究共鉴定出α7-nAChR亚基及另外120种蛋白。此外，在不表达α7-nAChR的样本中，还鉴定出369种可与α-银环蛇毒素结合的蛋白，由此明确了采用α-银环蛇毒素富集法开展α7-nAChR相关研究时的非特异性结合蛋白。相较于本团队此前采用α-银环蛇毒素亲和磁珠分离技术开展的α7-nAChR互作蛋白研究，本研究通过对比α7-nAChR特异性蛋白与α-银环蛇毒素结合蛋白的差异，优化了蛋白分离方法，并引入了当前最新的质谱分析技术。本研究鉴定得到的α7-nAChR相互作用组涵盖与α7-nAChR的表达、定位、功能或调控相关的各类蛋白，可为后续阐明这些相互作用如何参与人类疾病的发生发展提供重要研究基础。