Rodríguez Armando Alexei, Salceda Emilio, Garateix Anoland Georgina, Zaharenko André Junqueira, Peigneur Steve, López Omar, Pons Tirso, Richardson Michael, Díaz Maylín, Hernández Yasnay, Ständker Ludger, Tytgat Jan, Soto Enrique
Centro de Bioproductos Marinos (CEBIMAR), Loma y 37, Alturas del Vedado, CP 10400 Habana, Cuba.
Instituto de Fisiología, Universidad Autónoma de Puebla, 14 sur 6301, CU, San Manuel, Puebla, Puebla CP 72750, Mexico.
Peptides. 2014 Mar;53:3-12. doi: 10.1016/j.peptides.2013.06.003. Epub 2013 Jun 10.
Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.
海葵能产生具有药理学和生物医学研究价值的离子通道肽毒素。然而，作用于配体门控离子通道的肽，包括酸敏感离子通道（ASIC）毒素，仍未得到充分研究。PhcrTx1是从十字海葵（Phymanthus crucifer）中鉴定出的首个化合物，它是一种新型的ASIC抑制剂。该肽通过凝胶过滤、离子交换和反相色谱法进行纯化，随后使用膜片钳技术对分离的大鼠背根神经节（DRG）神经元的离子通道进行生物学评估。PhcrTx1能部分抑制ASIC电流（IC50约为100 nM），也能抑制电压门控钾离子电流，但在DRG神经元中，当浓度处于微摩尔范围时，其对峰值电流和稳态电流的影响低于20%。在DRG神经元中，未观察到对钠电压门控电流有显著影响。通过N端测序得到了32个氨基酸残基，质谱分析其分子量为3477 Da。未发现与其他海葵肽有序列同源性。有趣的是，对其半胱氨酸模式和二级结构排列的生物信息学分析表明，该肽呈现出抑制性胱氨酸结（ICK）支架结构，这种结构在蜘蛛、蝎子和芋螺等其他有毒生物中也有发现。我们的结果表明，PhcrTx1代表了作用于ASIC且对Kv通道作用效力低得多的海葵毒素新结构组的首个成员。此外，这是刺胞动物中ICK肽的首次报道，表明该基序在有毒动物中的出现比预期更为古老。
