Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni
Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni
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DOI：
10.1371/journal.ppat.1003586
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发表时间：
2013-08-01
期刊：
影响因子：
6.7
通讯作者：
Geary, Timothy G.
中科院分区：
文献类型：
--
作者：
Dufour, Vanessa;Beech, Robin N.;Geary, Timothy G.
Cys环配体门控离子通道（LGIC）介导快速离子神经传递。它们被证明是线虫和节肢动物中的药物靶标，但在扁虫中的特征很差。在这项研究中，我们的特点是阴离子选择性，非乙酰胆碱门控的半胱氨酸环LGIC曼氏血吸虫。获得SmGluCl-1（Smp_096480）、SmGluCl-2（Smp_015630）和SmGluCl-3（Smp_104890）的全长cDNA。检索SmGluCl-4的部分cDNA（Smp_099500/Smp_176730）。系统发育分析表明，SmGluCl-1，SmGluCl-2，SmGluCl-3和SmGluCl-4属于扁虫谷氨酸门控氯离子通道（GluCl）的一个新的分支，其中包括推定的基因吸虫和绦虫。扁形虫GluCl进化枝不同于线虫-节肢动物和软体动物GluCl进化枝，并且不同于所有GABA受体。我们在S. mansoni SmGluCl-1，SmGluCl-2和SmGluCl-3亚基的特点是在非洲爪蟾卵母细胞的双电极电压钳（TEVC），并显示编码氯渗透通道的谷氨酸门控。SmGluCl-2和SmGluCl-3产生功能性同聚体，而SmGluCl-1与SmGluCl-2形成异聚体。浓度-响应关系显示SmGluCl受体对L-谷氨酸的敏感性是GluCl受体中报道的最高的，EC 50值为7-26 μ M。氯离子选择性通过电流-电压（I/V）关系确认。SmGluCl受体对1 μ M伊维菌素（IVM）不敏感，表明它们不属于IVM高度敏感的GluCl α亚型组。SmGluCl受体也对10 μ M甲氯硝西泮（一种能杀死人体的苯二氮卓类药物）不敏感。这些结果提供了第一个分子证据，表明GluCl受体对S. mansoni，这是寄生扁形虫中前所未有的发现。需要进一步的工作，以阐明GluCl受体的作用，并探讨其作为药物靶点的潜力。
Cys-loop ligand-gated ion channels (LGICs) mediate fast ionotropic neurotransmission. They are proven drug targets in nematodes and arthropods, but are poorly characterized in flatworms. In this study, we characterized the anion-selective, non-acetylcholine-gated Cys-loop LGICs from Schistosoma mansoni. Full-length cDNAs were obtained for SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630) and SmGluCl-3 (Smp_104890). A partial cDNA was retrieved for SmGluCl-4 (Smp_099500/Smp_176730). Phylogenetic analyses suggest that SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 belong to a novel clade of flatworm glutamate-gated chloride channels (GluCl) that includes putative genes from trematodes and cestodes. The flatworm GluCl clade was distinct from the nematode-arthropod and mollusc GluCl clades, and from all GABA receptors. We found no evidence of GABA receptors in S. mansoni. SmGluCl-1, SmGluCl-2 and SmGluCl-3 subunits were characterized by two-electrode voltage clamp (TEVC) in Xenopus oocytes, and shown to encode Cl--permeable channels gated by glutamate. SmGluCl-2 and SmGluCl-3 produced functional homomers, while SmGluCl-1 formed heteromers with SmGluCl-2. Concentration-response relationships revealed that the sensitivity of SmGluCl receptors to L-glutamate is among the highest reported for GluCl receptors, with EC50 values of 7-26 mu M. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 mu M ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluCl alpha subtype group. SmGluCl receptors are also insensitive to 10 mu M meclonazepam, a schistosomicidal benzodiazepine. These results provide the first molecular evidence showing the contribution of GluCl receptors to L-glutamate signaling in S. mansoni, an unprecedented finding in parasitic flatworms. Further work is needed to elucidate the roles of GluCl receptors in schistosomes and to explore their potential as drug targets.