Paiva Ana L B, Matavel Alessandra, Peigneur Steve, Cordeiro Marta N, Tytgat Jan, Diniz Marcelo R V, de Lima Maria Elena
Departamento de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil; Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Departamento de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil.
Biochimie. 2016 Feb;121:326-35. doi: 10.1016/j.biochi.2015.12.019. Epub 2015 Dec 30.
The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl-d-aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P. nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50 = 212.5 nM), and at 1 μM caused an increase on current peak amplitude of 105.2 ± 3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43 ± 8.04%, IC50 = 1.5 μM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.
来自巴西游走蛛的毒素PnTx4(5 - 5)对昆虫具有极高的毒性/致死性，但脑内注射后在小鼠身上未观察到明显的行为影响。然而，已证明它能抑制培养的大鼠海马神经元中谷氨酸受体的N - 甲基 - d - 天冬氨酸（NMDA）亚型。PnTx4(5 - 5)与PnTx4(6 - 1)有63%的同源性，PnTx4(6 - 1)是来自巴西游走蛛的另一种杀虫毒素，它能减缓昆虫中枢神经系统中的钠电流失活，但对大鼠的Nav1.2和Nav1.4钠通道没有影响。在此，我们在大肠杆菌中克隆并异源表达了毒素PnTx4(5 - 5)。重组毒素rPnTx4(5 - 5)在德国小蠊的钠通道NavBg以及非洲爪蟾卵母细胞中表达的哺乳动物钠通道Nav1.2 - 1.6上进行了测试。我们发现该毒素对昆虫和哺乳动物钠通道具有不同的亲和力和作用方式。最显著的影响是对NavBg，rPnTx4(5 - 5)能强烈减缓通道失活（半数有效浓度EC50 = 212.5 nM），在1 μM时使电流峰值幅度增加105.2 ± 3.1%。有趣的是，该毒素也能抑制所有测试的哺乳动物通道上的钠电流，对Nav1.3的电流抑制作用更强（38.43 ± 8.04%，半数抑制浓度IC50 = 1.5 μM）。对Nav1.3和Nav1.5的激活曲线分析表明，该毒素使通道激活向更去极化的电位偏移，这可以解释钠电流的抑制现象。此外，该毒素也能轻微减缓Nav1.3和Nav1.6通道上的钠电流失活。据我们所知，这是首次描述的一种能使钠通道激活向更去极化电位偏移并减缓通道失活的蜘蛛目毒素。
