The three-finger toxin MTα is a selective α2B-adrenoceptor antagonist
The three-finger toxin MTα is a selective α2B-adrenoceptor antagonist
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DOI：
10.1016/j.toxicon.2010.05.001
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发表时间：
2010-09-01
期刊：
影响因子：
2.8
通讯作者：
Nasman, Johnny
中科院分区：
文献类型：
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作者：
Koivula, Katja;Rondinelli, Sergio;Nasman, Johnny
毒蕈碱毒素 (MT) 是从曼巴蛇毒液中分离出来的三指折叠肽。在本报告中，我们描述了 MT α 与人 α(2B)-肾上腺素受体的选择性拮抗相互作用。在功能测定中，测量 α(2B)-肾上腺素受体诱导的细胞内 [Ca2+] 增加，我们发现有毒 MT α 和合成 MT α 均以浓度依赖性方式抑制反应。 MT α 不影响 α(2A)、α(2c)、α(1A) 或 α(1B) 肾上腺素受体的反应。为了进一步探索 MT α 与 α(2B)-肾上腺素受体的结合，我们以 [H-3]RX821002 作为报告配体对 Sf9 细胞匀浆进行配体结合实验。 MT α 与受体的结合相当缓慢，需要大约 60 分钟才能达到平衡。在平衡结合实验中，MT α 以 3.2 nM 的 IC50 置换放射性配体，但不能置换所有结合的放射性配体。使用饱和结合方案，我们发现 MT α 抑制了最大结合，而对放射性配体的亲和力没有任何更大的影响，表明非竞争性抑制模式。该毒素可逆地与 α(2B)-肾上腺素受体结合，但需要大量清洗才能在高毒素浓度下完全恢复结合位点。令人惊讶的是，在完全阻断 α(2B)-肾上腺素受体的浓度下，MT α 并不影响 [H-3]-N-甲基东莨菪碱与毒蕈碱受体亚型的结合，这表明该毒素是 α(2B)-肾上腺素受体比毒蕈碱受体更有效的拮抗剂。这些发现应该在选择性肾上腺素受体药物设计以及阐明 α(2)-肾上腺素受体生理学方面开辟新的观点。 (C) 2010 Elsevier Ltd. 保留所有权利。
Muscarinic toxins (MTs) are three-finger folded peptides isolated from mamba snake venoms. In this report we describe a selective antagonistic interaction of MT alpha with the human alpha(2B)-adrenoceptor. In a functional assay, measuring the alpha(2B)-adrenoceptor-induced increase in intracellular [Ca2+], we found that both venomous MT alpha and synthetic MT alpha inhibited the response in a concentration-dependent way. MT alpha did not affect the responses of alpha(2A)-, alpha(2c)-, alpha(1A)- or alpha(1B)-adrenoceptors. To further explore the binding of MT alpha to the alpha(2B)-adrenoceptor, we performed ligand binding experiments on Sf9 cell homogenates with [H-3]RX821002 as reporter ligand. MT alpha bound to the receptor rather slowly requiring about 60 min to reach equilibrium. In equilibrium binding experiments, MT alpha displaced the radioligand with an IC50 of 3.2 nM, but was not able to displace all bound radioligand. Using a saturation binding protocol, we found that MT alpha suppressed the maximum binding without any greater impact on the affinity of the radioligand, indicating a non-competitive mode of inhibition. The toxin bound reversibly to alpha(2B)-adrenoceptor, but extensive washing was needed for full recovery of binding sites at high toxin concentrations. Surprisingly, MT alpha did not affect [H-3]-N-methylscopolamine binding to the muscarinic receptor subtypes at concentrations found to fully block alpha(2B)-adrenoceptors, showing that the toxin is a more potent antagonist for the alpha(2B)-adrenoceptor than for muscarinic receptors. These findings should open up new views in terms of selective adrenoceptor drug design as well as in elucidation of alpha(2)-adrenoceptor physiology. (C) 2010 Elsevier Ltd. All rights reserved.