Pharmacological properties of α9α10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras
Pharmacological properties of α9α10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras
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DOI：
10.1124/mol.65.2.453
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发表时间：
2004-02-01
影响因子：
3.6
通讯作者：
Millar, NS
中科院分区：
文献类型：
--
作者：
Baker, ER;Zwart, R;Millar, NS
烟碱乙酰胆碱受体（nAChR）α 9和α 10亚基主要在内耳的毛细胞内表达，并与听觉处理有关。虽然先前已经描述了通过在非洲爪蟾卵母细胞中共表达α 9和α 10产生的功能性重组nAChRs，但是还没有关于在培养的细胞系中成功异源表达α 9 α 10 nAChRs的报道。在本研究中，构建了亚基嵌合体（alpha 9 chi和alpha 10 chi），其含有与5-羟色胺3A型（5 HT（3A））亚基的C-末端结构域融合的alpha 9或alpha 10亚基的胞外配体结合结构域。在单独用α 9 chi或α 10 chi转染的哺乳动物细胞的膜制备物中检测到烟碱放射性配体[（3）H]甲基牛烟碱的特异性高亲和力结合，但当α 9 chi和α 10 chi共转染时检测到显著更高水平的结合，提供了需要α 9和α 10共组装以有效形成烟碱结合位点的证据。通过平衡放射性配体结合研究确定的alpha 9 alpha 10 chi受体的药理学特征与先前通过天然和重组alpha 9 alpha 10 nAChR进行的电生理学研究确定的药理学特征大致相似。与α 9 α 10 nAChR表现出非典型药理学特征的证据一致，我们已经鉴定了几种非烟碱配体的特异性高亲和力结合，包括士的宁（甘氨酸受体拮抗剂）、荷包牡丹碱（GABA（A）受体拮抗剂）和阿托品（毒蕈碱乙酰胆碱受体拮抗剂）。结果还与用先前描述的α 7/5 HT（3A）（α 7 chi）亚基嵌合体进行的放射性配体结合数据进行了比较。
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 (5HT(3A)) subunit. Specific high-affinity binding of the nicotinic radioligand [(3)H] 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 alpha9alpha10chi 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 GABA(A) receptor antagonist), and atropine ( a muscarinic acetylcholine receptor antagonist). Results have also been compared with radioligand binding data conducted with a previously described alpha7/5HT(3A) (alpha7chi) subunit chimera.