López-Vera Estuardo, Aguilar Manuel B, Schiavon Emanuele, Marinzi Chiara, Ortiz Ernesto, Restano Cassulini Rita, Batista Cesar V F, Possani Lourival D, Heimer de la Cotera Edgar P, Peri Francesco, Becerril Baltazar, Wanke Enzo
Laboratorio de Neurofarmacología Marina, Departamento de Neurobiología Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Campus Juriquilla, Queretaro, México.
FEBS J. 2007 Aug;274(15):3972-85. doi: 10.1111/j.1742-4658.2007.05931.x. Epub 2007 Jul 16.
alpha-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we describe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Channel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4/7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [gamma15E]SrIB) were compared to those exerted by the alpha4/7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing alpha(1)beta(1)gammadelta, alpha(4)beta(2) and alpha(3)beta(4) nicotinic acetylcholine receptors. At high concentrations (10 microm), the peptides SrIA, SrIB and [gamma15E]SrIB showed weak blocking effects only on alpha(4)beta(2) and alpha(1)beta(1)gammadelta subtypes, but EI also strongly blocked alpha(3)beta(4) receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors if briefly (2-15 s) applied at concentrations several orders of magnitude lower (EC(50), 1.78 and 0.37 nm, respectively). These results suggest not only that the novel alpha-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both alpha(1)beta(1)gammadelta and alpha(4)beta(2) nicotinic acetylcholine receptors with very high affinity and increase their intrinsic cholinergic response. Their unique properties make them excellent tools for studying the toxin-receptor interaction, as well as models with which to design highly specific therapeutic drugs.
海洋蜗牛中的α-芋螺毒素是已知的烟碱型乙酰胆碱受体的选择性强效竞争性拮抗剂。在此，我们描述了从采集于墨西哥尤卡坦海峡的刺芋螺中分离出的两种新型毒素SrIA和SrIB的纯化、结构特征及活性。通过直接氨基酸和cDNA核苷酸测序确定，这些毒素是含有18个氨基酸残基的肽，具有典型的4/7型框架，但序列全新。因此，将它们（以及一种合成类似物[γ15E]SrIB）的作用与用作对照的来自ermineus芋螺的α4/7-芋螺毒素EI的作用进行了比较。通过膜片钳技术在表达α(1)β(1)γδ、α(4)β(2)和α(3)β(4)烟碱型乙酰胆碱受体的哺乳动物细胞中评估了它们的靶标特异性。在高浓度（10微摩尔）下，肽SrIA、SrIB和[γ15E]SrIB仅对α(4)β(2)和α(1)β(1)γδ亚型表现出微弱的阻断作用，但EI也强烈阻断α(3)β(4)受体。与这种阻断作用相反，如果以低几个数量级的浓度（EC50分别为1.78和0.37纳米）短暂（2 - 15秒）施加，新肽和EI对α(1)β(1)γδ和α(4)β(2)烟碱型乙酰胆碱受体表现出显著的增强作用。这些结果不仅表明新型α-芋螺毒素和EI可作为烟碱型乙酰胆碱受体抑制剂发挥作用，还表明它们以非常高的亲和力结合α(1)β(1)γδ和α(4)β(2)烟碱型乙酰胆碱受体并增加其内在胆碱能反应。它们的独特性质使其成为研究毒素 - 受体相互作用的优秀工具，以及设计高度特异性治疗药物的模型。
