Baker Elizabeth R, Zwart Ruud, Sher Emanuele, Millar Neil S
Department of Pharmacology, University College London, Gower Street, London, United Kingdom.
Mol Pharmacol. 2004 Feb;65(2):453-60. doi: 10.1124/mol.65.2.453.
The nicotinic acetylcholine receptor (nAChR) alpha9 and alpha10 subunits are expressed primarily within hair cells of the inner ear and have been implicated in auditory processing. Although functional recombinant nAChRs generated by the coexpression of alpha9 and alpha10 in Xenopus laevis oocytes have been described previously, there have been no reports of the successful heterologous expression of alpha9alpha10 nAChRs in cultured cell lines. In this study, subunit chimeras (alpha9chi and alpha10chi) have been constructed that contain the extracellular, ligand binding domain of the alpha9 or alpha10 subunits fused to the C-terminal domain of the 5-hydroxytryptamine type 3A (5HT3A) subunit. Specific high-affinity binding of the nicotinic radioligand [3H]methyllycaconitine was detected in membrane preparations of mammalian cells transfected with alpha9chi or alpha10chi alone, but significantly higher levels of binding were detected when alpha9chi and alpha10chi were cotransfected, providing evidence of a requirement for coassembly of alpha9 and alpha10 for the efficient formation of a nicotinic binding site. The pharmacological profile of alpha9chialpha10chi receptors, determined by equilibrium radioligand binding studies, is broadly similar to that determined previously by electrophysiological studies conducted with native and recombinant alpha9alpha10 nAChRs. In agreement with evidence that alpha9alpha10 nAChRs exhibit an atypical pharmacological profile, we have identified specific high-affinity binding of several non-nicotinic ligands including strychnine (a glycine receptor antagonist), bicuculline (a GABAA receptor antagonist), and atropine (a muscarinic acetylcholine receptor antagonist). Results have also been compared with radioligand binding data conducted with a previously described alpha7/5HT3A (alpha7chi) subunit chimera.
烟碱型乙酰胆碱受体（nAChR）α9和α10亚基主要在内耳毛细胞中表达，并与听觉处理有关。尽管此前已描述了通过在非洲爪蟾卵母细胞中共表达α9和α10产生的功能性重组nAChR，但尚无关于α9α10 nAChR在培养细胞系中成功异源表达的报道。在本研究中，构建了亚基嵌合体（α9chi和α10chi），其包含α9或α10亚基的细胞外配体结合结构域，与5-羟色胺3A（5HT3A）亚基的C末端结构域融合。在用单独的α9chi或α10chi转染的哺乳动物细胞膜制剂中检测到了烟碱型放射性配体[3H]甲基lycaconitine的特异性高亲和力结合，但当α9chi和α10chi共转染时检测到的结合水平明显更高，这为α9和α10共同组装以有效形成烟碱型结合位点提供了证据。通过平衡放射性配体结合研究确定的α9chialpha10chi受体的药理学特征与先前通过对天然和重组α9α10 nAChR进行电生理研究确定的特征大致相似。与α9α10 nAChR表现出非典型药理学特征的证据一致，我们鉴定了几种非烟碱型配体的特异性高亲和力结合，包括士的宁（一种甘氨酸受体拮抗剂）、荷包牡丹碱（一种GABAA受体拮抗剂）和阿托品（一种毒蕈碱型乙酰胆碱受体拮抗剂）。还将结果与先前描述的α7/5HT3A（α7chi）亚基嵌合体的放射性配体结合数据进行了比较。
