Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor
Interaction of α-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor
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DOI：
10.1111/j.1471-4159.2009.06359.x
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发表时间：
2009-11-01
影响因子：
4.7
通讯作者：
Tsetlin, Victor I.
中科院分区：
文献类型：
--
作者：
Kasheverov, Igor E.;Zhmak, Maxim N.;Tsetlin, Victor I.
α-芋螺毒素在激动剂/竞争性拮抗剂的位点与烟碱乙酰胆碱受体（nAChR）和乙酰胆碱结合蛋白（AChBP）相互作用。阻断肌肉型或α 7 nAChR的α-芋螺毒素与α-银环蛇毒素竞争。然而，α-芋螺毒素ImII（α 7 nAChR靶向α-芋螺毒素ImI的密切同源物）阻断α 7和肌肉nAChR而不置换α-银环蛇毒素（Ellison等人，2003，2004），表明在不同位点结合。我们合成了α-芋螺毒素ImII，它的带状异构体（ImIIiso），'突变体' ImII（W10 Y），并发现在阻断人类α 7和肌肉nAChR在非洲爪蟾卵母细胞中的类似效力。这两种异构体都能从细胞系GH（4）C（1）中的人α 7 nAChR（IC 50分别为17和23 μ M）以及从滞水蛙和加利福尼亚蛙AChBP（IC 50为2.0-9.0 μ M）中置换[125 I]-α-银环蛇毒素。根据SPR测量，这两种异构体结合到固定的AChBP和AChBP竞争固定的α-银环蛇毒素（Kd和IC 50 2.5-8.2 μ M）。α-芋螺毒素[125 I]-ImII（W10 Y）对电鳐nAChR显示特异性结合（Kd 1.5-6.1 μ M），并可被α-芋螺毒素ImII、ImIIiso和ImII（W10 Y）取代，IC 50分别为2.7、2.2和3.1 μ M。由于α-眼镜蛇毒素和α-芋螺毒素ImI仅在较高浓度（IC 50>= 90 μ M）下取代[125 I]-ImII（W10 Y），我们的结果表明α-芋螺毒素ImII及其同系物在电鳐nAChR上具有不同于激动剂/竞争性拮抗剂的额外结合位点。
alpha-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. alpha-Conotoxins blocking muscle-type or alpha 7 nAChRs compete with alpha-bungarotoxin. However, alpha-conotoxin ImII, a close homolog of the alpha 7 nAChR-targeting alpha-conotoxin ImI, blocked alpha 7 and muscle nAChRs without displacing alpha-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized alpha-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human alpha 7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [125I]-alpha-bungarotoxin from human alpha 7 nAChRs in the cell line GH(4)C(1) (IC50 17 and 23 mu M, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC50 2.0-9.0 mu M). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized alpha-bungarotoxin (K-d and IC50 2.5-8.2 mu M). On Torpedo nAChR, alpha-conotoxin [125I]-ImII(W10Y) revealed specific binding (K-d 1.5-6.1 mu M) and could be displaced by alpha-conotoxin ImII, ImIIiso and ImII(W10Y) with IC50 2.7, 2.2 and 3.1 mu M, respectively. As alpha-cobratoxin and alpha-conotoxin ImI displaced [125I]-ImII(W10Y) only at higher concentrations (IC50 >= 90 mu M), our results indicate that alpha-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists.