Pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 nicotinic acetylcholine receptors: subunit arrangement determines functional expression
Pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 nicotinic acetylcholine receptors: subunit arrangement determines functional expression
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DOI：
10.1111/j.1476-5381.2008.00104.x
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发表时间：
2009-03-01
影响因子：
7.3
通讯作者：
Bermudez, I.
中科院分区：
文献类型：
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作者：
Carbone, A-L;Moroni, M.;Bermudez, I.
在非洲爪蟾卵母细胞中异源表达的 α 4 和 β 2 烟碱乙酰胆碱 (ACh) 受体亚基组装成 (α 4)(2)(β 2)(3) 和 (α 4)(3)(β 2)(2) 受体的混合群体。为了在异源系统中分别表达这些受体，我们设计了五聚体串联的 (α 4)(2)(β 2)(3) 和 (α 4)(3)(β 2)(2) 受体。α 4 和 β 2 亚基通过合成接头连接成五聚体构建体，以产生 (α 4)(2)(β 2)(3) 或 (α 4)(3)(β) 2)(2)受体。使用两电极电压钳技术，我们检查了串联构建体在非洲爪蟾卵母细胞中产生功能表达的能力。进一步对功能构建体的激动剂、竞争性拮抗剂、Ca2+通透性、对Zn2+调节的敏感性和对伴侣蛋白14-3-3上调的敏感性进行了表征。我们发现具有β2_α4_β2_α4_β2或β2_α4_β2_α4_α4亚基排列的五聚体在非洲爪蟾卵母细胞中是稳定且有功能的。相比之下，当α 4和β 2与β 2_β 2_α 4_β 2_α 4或β 2_α 4_α 4_β 2_α 4的亚基顺序连接时，非洲爪蟾卵母细胞中的功能表达非常低，尽管蛋白质已合成且稳定。 beta 2_alpha 4_beta 2_alpha 4_beta 2 和 beta 2_alpha 4_beta 2_alpha 4_alpha 4 连接体均概括了 ACh 浓度响应曲线、对 Zn2+ 调节的敏感性、Ca2+ 通透性以及对相应非连接 (alpha 4)(2)(beta 2)(3) 和 (alpha) 的伴侣蛋白 14-3-3 上调的敏感性分别为4)(3)(β 2)(2)受体。使用这些多联体，我们发现大多数研究的 α 4 β 2 偏好化合物，包括 A85380、5I-A85380、金雀花碱、地巴替丁、TC2559 和二氢-β-赤霉素，均表现出化学计量特异性的效力和功效。我们得出结论，用 β 2_α 4_β 2_α 产生的 α 4 β 2 烟碱 ACh 受体4_beta 2 或 beta 2_alpha 4_beta 2_alpha 4_alpha 4 五聚体构建体分别是非连接的 (alpha 4)(2)(beta 2)(3) 和 (alpha 4)(3)(beta 2)(2) 受体的有效模型。
alpha 4 and beta 2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors.alpha 4 and beta 2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (alpha 4)(2)(beta 2)(3) or (alpha 4)(3)(beta 2)(2) receptors. Using two-electrode voltage-clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca2+ permeability, sensitivity to modulation by Zn2+ and sensitivity to up-regulation by chaperone protein 14-3-3.We found that pentameric concatamers with a subunit arrangement of beta 2_alpha 4_beta 2_alpha 4_beta 2 or beta 2_alpha 4_beta 2_alpha 4_alpha 4 were stable and functional in Xenopus oocytes. By comparison, when alpha 4 and beta 2 were concatenated with a subunit order of beta 2_beta 2_alpha 4_beta 2_alpha 4 or beta 2_alpha 4_alpha 4_beta 2_alpha 4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both beta 2_alpha 4_beta 2_alpha 4_beta 2 and beta 2_alpha 4_beta 2_alpha 4_alpha 4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn2+ modulation, Ca2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors respectively. Using these concatamers, we found that most alpha 4 beta 2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-beta-erythroidine, demonstrate stoichiometry-specific potencies and efficacies.We concluded that the alpha 4 beta 2 nicotinic ACh receptors produced with beta 2_alpha 4_beta 2_alpha 4_beta 2 or beta 2_alpha 4_beta 2_alpha 4_alpha 4 pentameric constructs are valid models of non-linked (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2) receptors respectively.