Moretti Milena, Zoli Michele, George Andrew A, Lukas Ronald J, Pistillo Francesco, Maskos Uwe, Whiteaker Paul, Gotti Cecilia
CNR Institute of Neuroscience, Biometra University of Milan, Milan, Italy (M.M., F.P., C.G.); Section of Physiology and Neurosciences, Department of Biomedical, Metabolic, and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy (M.Z.); Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (A.A.G., R.J.L., P.W.); and Centre National de la Recherche Scientifique, Unité Neurobiologie Intégrative des Systèmes Cholinergiques, Institut Pasteur, Paris, France (U.M.).
CNR Institute of Neuroscience, Biometra University of Milan, Milan, Italy (M.M., F.P., C.G.); Section of Physiology and Neurosciences, Department of Biomedical, Metabolic, and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy (M.Z.); Division of Neurobiology, Barrow Neurologic Institute, Phoenix, Arizona (A.A.G., R.J.L., P.W.); and Centre National de la Recherche Scientifique, Unité Neurobiologie Intégrative des Systèmes Cholinergiques, Institut Pasteur, Paris, France (U.M.)
Mol Pharmacol. 2014 Sep;86(3):306-17. doi: 10.1124/mol.114.093377. Epub 2014 Jul 7.
We examined α7β2-nicotinic acetylcholine receptor (α7β2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7β2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7β2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using β2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(β2)1-, and (α7)3(β2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7β2- versus α7-nAChRs. This study represents the first direct confirmation of α7β2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7β2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7β2-nAChR subunit isoforms with different α7/β2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7β2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.
我们检测了哺乳动物脑中α7β2-烟碱型乙酰胆碱受体（α7β2-nAChR）的表达，并比较了同源α7-nAChR和α7β2-nAChR的药理学特性。使用α-银环蛇毒素亲和纯化或用抗α7亚基抗体（Abs）进行免疫沉淀，从小鼠或人脑样本中分离出含有α7亚基的nAChR。基于使用β2亚基特异性Abs对分离物进行的蛋白质印迹分析，在两个物种的前脑中均检测到α7β2-nAChR，但在其他测试区域未检测到。在使用亚基缺失突变小鼠或异源表达特定人类nAChR亚型和亚基的细胞系的对照研究中证实了抗体的特异性。使用双电极电压钳记录对烟碱配体的反应，证实了串联五聚体（α7）5-、（α7）4（β2）1-和（α7）3（β2）2-nAChR在非洲爪蟾卵母细胞中的功能性表达。重要的是，串联（α7）5-nAChR或由未连接的α7亚基构成的同源α7-nAChR的药理学特性无法区分。（α7）5-、（α7）4（β2）1-和（α7）3（β2）2-nAChR的药理学特性相似，只是与α7-nAChR相比，α7β2-nAChR上尼古丁的效力（相对于乙酰胆碱效力进行归一化）有所降低。这项研究首次直接证实了α7β2-nAChR在人和小鼠前脑中的表达，支持了先前关于α7β2-nAChR与阿尔茨海默病发病机制相关性的小鼠研究。这些数据还表明，具有不同α7/β2亚基比例的α7β2-nAChR亚基异构体彼此之间以及与α7同五聚体nAChR具有相似的药理学特性。这支持了以下假设：α7β2-nAChR激动剂激活主要或完全反映与α7/α7亚基界面位点的结合。
