Molecular Cloning and Characterization of Novel Glutamate-Gated Chloride Channel Subunits from Schistosoma mansoni

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 µM. Chloride selectivity was confirmed by current-voltage (I/V) relationships. SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group. SmGluCl receptors are also insensitive to 10 µM 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.半胱氨酸环配体门控离子通道（Cys-loop ligand-gated ion channels, LGICs）可介导快速离子型神经传递。该类通道已被证实为线虫与节肢动物的药物靶点，但在扁形动物中的功能特征仍有待深入解析。本研究针对曼氏血吸虫（Schistosoma mansoni）中的阴离子选择性非乙酰胆碱门控型半胱氨酸环LGICs展开功能特征分析。 我们成功获取了SmGluCl-1（Smp_096480）、SmGluCl-2（Smp_015630）与SmGluCl-3（Smp_104890）的全长互补DNA（complementary DNA, cDNA），并获得了SmGluCl-4（Smp_099500/Smp_176730）的部分cDNA序列。 系统发育分析结果显示，SmGluCl-1、SmGluCl-2、SmGluCl-3与SmGluCl-4隶属于一个全新的扁形动物谷氨酸门控氯离子通道（glutamate-gated chloride channel, GluCl）进化支，该进化支包含吸虫纲与绦虫纲的推定功能基因。扁形动物GluCl进化支与线虫-节肢动物、软体动物GluCl进化支及所有γ-氨基丁酸（GABA）受体均存在显著差异，且本研究未在曼氏血吸虫中检测到GABA受体的存在证据。 我们通过非洲爪蟾卵母细胞双电极电压钳（two-electrode voltage clamp, TEVC）对SmGluCl-1、SmGluCl-2与SmGluCl-3亚基进行功能表征，证实其可编码谷氨酸门控的氯离子选择性通道。其中，SmGluCl-2与SmGluCl-3可形成功能性同源多聚体，而SmGluCl-1则需与SmGluCl-2结合形成异源多聚体方可行使功能。浓度-反应关系实验结果表明，SmGluCl受体对L-谷氨酸的敏感性处于已报道的GluCl受体中的较高水平，半数有效浓度（median effective concentration, EC50）介于7~26 μM之间。研究通过电流-电压（I/V）关系实验进一步验证了该受体的氯离子选择性。 SmGluCl受体对1 μM伊维菌素（ivermectin, IVM）不敏感，提示其不属于对伊维菌素高度敏感的GluClα亚型家族。此外，该受体对10 μM美克洛奈泮——一种抗血吸虫苯二氮䓬类药物——同样不敏感。 本研究首次从分子层面证实了GluCl受体参与曼氏血吸虫的L-谷氨酸信号传导过程，这一发现于寄生扁形动物中尚属首次。后续仍需开展进一步研究，以阐明GluCl受体在血吸虫体内的具体生物学功能，并探索其作为药物靶点的潜在应用价值。