α-Conotoxins Identify the α3β4☆ Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells
α-Conotoxins Identify the α3β4☆ Subtype as the Predominant Nicotinic Acetylcholine Receptor Expressed in Human Adrenal Chromaffin Cells
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DOI：
10.1124/mol.115.100982
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发表时间：
2015-11-01
影响因子：
3.6
通讯作者：
Albillos, Almudena
中科院分区：
文献类型：
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作者：
Hone, Arik J.;McIntosh, J. Michael;Albillos, Almudena
缺乏选择性抑制人α 3 β 2和α 6 β 2烟碱乙酰胆碱受体（nAChR）而不是密切相关的α 3 β 4和α 6 β 4亚型的配体。目前的α-芋螺毒素（α-Ctx），区分这些nAChR亚型在大鼠之间不能区分人类受体同源物。在这项研究中，我们描述了α-Ctx LvIA（N9 R，V10 A）的开发，其对卵母细胞表达的人α 3 β 2的效力是α 3 β 4的3000倍，对人α 6/α 3 β 2 β 3的效力是α 6/α 3 β 4 nAChR的165倍。该类似物与其他三种α-Ctx类似物和膜片钳电生理学一起用于表征人肾上腺嗜铬细胞表达的nAChR亚型。LvIA（N9 R，V10 A）在预期抑制具有β 2配体结合位点的nAChR的浓度下对这些细胞中的乙酰胆碱诱发电流几乎没有影响。相比之下，β 4-选择性α-Ctx BuIA（T5 A，P6 O）抑制>98%的乙酰胆碱诱发电流，表明大多数异聚体受体含有β 4配体结合位点。使用α 6选择性α-Ctx PeIA（A7 V、S9 H、V10 A、N11 R、E14 A）的其他研究表明，人肾上腺嗜铬细胞表达的主要异聚nAChR是α 3 β 4* 亚型（星号表示可能存在其他亚基）。这一结论得到了人肾上腺髓质和培养的人肾上腺嗜铬细胞的聚合酶链反应实验的支持，这些实验表明α 3、α 5、α 7、β 2和β 4亚基的RNA表达显著，α 2、α 4、α 6和α 10亚基的RNA丰度较低。
Ligands that selectively inhibit human alpha 3 beta 2 and alpha 6 beta 2 nicotinic acetylcholine receptor (nAChRs) and not the closely related alpha 3 beta 4 and alpha 6 beta 4 subtypes are lacking. Current alpha-conotoxins (alpha-Ctxs) that discriminate among these nAChR subtypes in rat fail to discriminate among the human receptor homologs. In this study, we describe the development of alpha-Ctx LvIA(N9R, V10A) that is 3000-fold more potent on oocyte-expressed human alpha 3 beta 2 than alpha 3 beta 4 and 165-fold more potent on human alpha 6/alpha 3 beta 2 beta 3 than alpha 6/alpha 3 beta 4 nAChRs. This analog was used in conjuction with three other alpha-Ctx analogs and patch-clamp electrophysiology to characterize the nAChR subtypes expressed by human adrenal chromaffin cells. LvIA(N9R, V10A) showed little effect on the acetylcholine-evoked currents in these cells at concentrations expected to inhibit nAChRs with beta 2 ligand-binding sites. In contrast, the beta 4-selective alpha-Ctx BuIA(T5A, P6O) inhibited >98% of the acetylcholine-evoked current, indicating that most of the heteromeric receptors contained beta 4 ligand-binding sites. Additional studies using the alpha 6-selective alpha-Ctx PeIA(A7V, S9H, V10A, N11R, E14A) indicated that the predominant heteromeric nAChR expressed by human adrenal chromaffin cells is the alpha 3 beta 4* subtype (asterisk indicates the possible presence of additional subunits). This conclusion was supported by polymerase chain reaction experiments of human adrenal medulla gland and of cultured human adrenal chromaffin cells that demonstrated prominent expression of RNAs for alpha 3, alpha 5, alpha 7, beta 2, and beta 4 subunits and a low abundance of RNAs for alpha 2, alpha 4, alpha 6, and alpha 10 subunits.