Nicotinic acetylcholine receptors (nAChRs) are pentameric ion channels expressed in the central nervous systems. nAChRs containing the α4, β2 and α5 subunits are specifically involved in addictive processes, but their functional architecture is poorly understood due to the intricacy of assembly of these subunits. Here we constrained the subunit assembly by designing fully concatenated human α4β2 and α4β2α5 receptors and characterized their properties by two-electrodes voltage–clamp electrophysiology in Xenopus oocytes. We found that α5-containing nAChRs are irreversibly blocked by methanethiosulfonate (MTS) reagents through a covalent reaction with a cysteine present only in α5. MTS-block experiments establish that the concatemers are expressed in intact form at the oocyte surface, but that reconstitution of nAChRs from loose subunits show inefficient and highly variable assembly of α5 with α4 and β2. Mutational analysis shows that the concatemers assemble both in clockwise and anticlockwise orientations, and that α5 does not contribute to ACh binding from its principal (+) site. Reinvestigation of suspected α5-ligands such as galantamine show no specific effect on α5-containing concatemers. Analysis of the α5-D398N mutation that is linked to smoking and lung cancer shows no significant effect on the electrophysiological function, suggesting that its effect might arise from alteration of other cellular processes. The concatemeric strategy provides a well-characterized platform for mechanistic analysis and screening of human α5-specific ligands.

烟碱乙酰胆碱受体 (nAChR) 是在中枢神经系统中表达的五聚体离子通道。含有α4、β2和α5亚基的nAChR专门参与成瘾过程，但由于这些亚基组装的复杂性，人们对它们的功能结构知之甚少。在这里，我们通过设计完全串联的人类α4β2和α4β2α5受体来限制亚基组装，并通过爪蟾卵母细胞中的双电极电压钳电生理学来表征它们的特性。我们发现，含有 α5 的 nAChR 通过与仅存在于 α5 中的半胱氨酸发生共价反应，被甲硫代磺酸盐 (MTS) 试剂不可逆地阻断。 MTS 块实验证实，多联体在卵母细胞表面以完整形式表达，但从松散亚基重建 nAChR 显示 α5 与 α4 和 β2 的组装效率低且高度可变。突变分析表明，多联体以顺时针和逆时针方向组装，并且 α5 对其主 (+) 位点不参与 ACh 结合。对可疑 α5 配体（例如加兰他敏）的重新研究显示，对含 α5 的多联体没有特定作用。对与吸烟和肺癌相关的α5-D398N突变的分析显示，对电生理功能没有显着影响，表明其影响可能来自其他细胞过程的改变。串联策略为人类 α5 特异性配体的机制分析和筛选提供了一个特征良好的平台。