Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists
Stable expression and functional characterization of a human nicotinic acetylcholine receptor with α6β2 properties: discovery of selective antagonists
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DOI：
10.1111/j.1476-5381.2011.01213.x
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发表时间：
2011-05-01
影响因子：
7.3
通讯作者：
Mugnaini, Manolo
中科院分区：
文献类型：
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作者：
Capelli, Anna Maria;Castelletti, Laura;Mugnaini, Manolo
背景和技术尽管越来越多的证据表明抑制含α 6 β 2（α 6 β 2*）的烟碱乙酰胆碱受体（nAChR）可能有益于烟草成瘾的治疗，但缺乏α 6 β 2*-nAChR的良好来源已经延迟了α 6 β 2-选择性拮抗剂的发现。我们的目的是产生稳定表达具有α 6 β 2特性的功能性nAChR的细胞系，以能够进行药理学表征和鉴定新的α 6 β 2-选择性拮抗剂。将嵌合α 6/3和突变β 3V 273 S亚基转染到人胚肾细胞中，并在荧光成像板读数器测定中测试其活性。大鼠免疫固定α 6 β 2 ~*-nAChRs的药理学研究表明，α 6/3、β 2和β 3V 273 S亚基共转染后，检测到功能性通道，而所有其他亚基组合均不能产生激动剂诱导的反应。稳定表达的α 6/3 β 2 β 3V 273 S-nAChR药理学是独特的，并且明显不同于α 4 β 2-、α 3 β 4-、α 7-和α 1 β 1 δ ε-nAChR。拮抗剂抑制α 6/3 β 2 β 3V 273 S-nAChR的效力与其对大鼠天然α 6 β 2*-nAChR的结合亲和力相似。激动剂对α 6 β 2*-nAChR的亲和力高于其激活α 6/3 β 2 β 3V 273 S-nAChR的效力，但其相对活性相当。在α 6/3 β 2 β 3V 273 S-nAChRs上进行集中筛选，然后与其他nAChRs交叉筛选，导致鉴定新的α 6 β 2选择性拮抗剂。结论和意义我们产生了一种稳定表达nAChRs的哺乳动物细胞系，其药理学性质类似于天然α 6 β 2*-nAChRs，并用它鉴定新的非肽，低分子量，α 6 β 2选择性拮抗剂。我们还提出了α 6 β 2拮抗剂的药效团模型，这为开发新的戒烟药物提供了一个起点。
BACKGROUND AND PURPOSEDespite growing evidence that inhibition of alpha 6 beta 2-containing (alpha 6 beta 2*) nicotinic acetylcholine receptors (nAChRs) may be beneficial for the therapy of tobacco addiction, the lack of good sources of alpha 6 beta 2*-nAChRs has delayed the discovery of alpha 6 beta 2-selective antagonists. Our aim was to generate a cell line stably expressing functional nAChRs with alpha 6 beta 2 properties, to enable pharmacological characterization and the identification of novel alpha 6 beta 2-selective antagonists.EXPERIMENTAL APPROACHDifferent combinations of the alpha 6, beta 2, beta 3, chimeric alpha 6/3 and mutant beta 3V273S subunits were transfected in human embryonic kidney cells and tested for activity in a fluorescent imaging plate reader assay. The pharmacology of rat immune-immobilized alpha 6 beta 2*-nAChRs was determined with 125I-epibatidine binding.KEY RESULTSFunctional channels were detected after co-transfection of alpha 6/3, beta 2 and beta 3V273S subunits, while all other subunit combinations failed to produce agonist-induced responses. Stably expressed alpha 6/3 beta 2 beta 3V273S-nAChR pharmacology was unique, and clearly distinct from alpha 4 beta 2-, alpha 3 beta 4-, alpha 7- and alpha 1 beta 1 delta epsilon-nAChRs. Antagonist potencies in inhibiting alpha 6/3 beta 2 beta 3V273S-nAChRs was similar to their binding affinity for rat native alpha 6 beta 2*-nAChRs. Agonist affinities for alpha 6 beta 2*-nAChRs was higher than their potency in activating alpha 6/3 beta 2 beta 3V273S-nAChRs, but their relative activities were equivalent. Focussed set screening at alpha 6/3 beta 2 beta 3V273S-nAChRs, followed by cross-screening with the other nAChRs, led to the identification of novel alpha 6 beta 2-selective antagonists.CONCLUSIONS AND IMPLICATIONSWe generated a mammalian cell line stably expressing nAChRs, with pharmacological properties similar to native alpha 6 beta 2*-nAChRs, and used it to identify novel non-peptide, low molecular weight, alpha 6 beta 2-selective antagonists. We also propose a pharmacophore model of alpha 6 beta 2 antagonists, which offers a starting point for the development of new smoking cessation agents.