Clostridium perfringens epsilon toxin (ETX) is a heptameric pore-forming toxin of the aerolysin toxin family. ETX is the most potent toxin of this toxin family and the third most potent bacterial toxin with high cytotoxic and lethal activities in animals. In addition, ETX shows a demyelinating activity in nervous tissue leading to devastating multifocal central nervous system white matter disease in ruminant animals. Pore formation in target cell membrane is most likely the initial critical step in ETX biological activity. Eight single to quadruple ETX mutants were generated by replacement of polar residues (serine, threonine, glutamine) in middle positions of the β-strands forming the β-barrel and facing the channel lumen with charged glutamic residues. Channel activity and ion selectivity were monitored in artificial lipid monolayer membranes and cytotoxicity was investigated in MDCK cells by the viability MTT test and propidium iodide entry. All the mutants formed channels with similar conductance in artificial lipid membranes and increasing cation selectivity for increasing number of mutations. Here, we show that residues in the central position of each β-strand of the amphipathic β-hairpin loop that forms the transmembrane pore, control the size and ion selectivity of the channel. While the highest cationic ETX mutants were not cytotoxic, no strict correlation was observed between ion selectivity and cytotoxicity.

产气荚膜梭菌ε毒素（ETX）是气溶素毒素家族的七聚体成孔毒素。ETX是该毒素家族中最有效的毒素，并且是在动物中具有高细胞毒性和致死性的第三大有效细菌毒素。此外，ETX在神经组织中表现出脱髓鞘活性，从而导致反刍动物体内毁灭性的多灶性中枢神经系统白质病。靶细胞膜中的孔形成很可能是ETX生物活性中的关键关键步骤。通过置换形成β桶的β链中间位置的极性残基（丝氨酸，苏氨酸，谷氨酰胺）产生八个八个ETX突变体，其中带电荷的谷氨酸残基面对通道管腔。在人工脂质单层膜中监测通道活性和离子选择性，并通过MTT活性测试和碘化丙锭进入研究MDCK细胞的细胞毒性。所有突变体均在人工脂质膜上形成了具有相似电导率的通道，并为增加突变数量而增加了阳离子选择性。在这里，我们显示了形成跨膜孔的两亲性β-发夹环的每个β链中心位置的残基，可控制通道的大小和离子选择性。虽然最高的阳离子ETX突变体没有细胞毒性，但在离子选择性和细胞毒性之间未观察到严格的相关性。所有突变体均在人工脂质膜上形成了具有相似电导率的通道，并为增加突变数量而增加了阳离子选择性。在这里，我们显示了形成跨膜孔的两亲性β-发夹环的每个β链中心位置的残基，可控制通道的大小和离子选择性。虽然最高的阳离子ETX突变体没有细胞毒性，但在离子选择性和细胞毒性之间未观察到严格的相关性。所有突变体均在人工脂质膜上形成了具有相似电导率的通道，并为增加突变数量而增加了阳离子选择性。在这里，我们显示了形成跨膜孔的两亲性β-发夹环的每个β链中心位置的残基，可控制通道的大小和离子选择性。虽然最高的阳离子ETX突变体没有细胞毒性，但在离子选择性和细胞毒性之间未观察到严格的相关性。