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

Abstract

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 EC ₅₀ 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.

Author Summary

Schistosomiasis is a debilitating disease caused by blood flukes in the genus Schistosoma that afflicts over 200 million people worldwide. Treatment relies almost exclusively on a single drug, praziquantel. Reports of sub-optimal efficacy of praziquantel raise concerns about the prospect of drug resistance and highlight the need to develop new schistosomicidal drugs. Neuroactive receptors are recognized targets of insecticides and anthelmintics. Likewise, neuronal receptors of schistosomes are attractive targets for drug development. Lacking a coelom and a proper circulatory system, schistosomes are thought to lack the capacity for endocrine signaling, and therefore depend entirely on neuronal modulation to control functions vital to their survival and reproduction. We characterized a novel family of glutamate-gated chloride channel (GluCl) receptors from S. mansoni that are pharmacologically and evolutionarily distinct from GluCls in nematodes, insects and snails. Our phylogenetic analyses suggest that these receptors are also widely distributed in other flukes and tapeworms. This study provides the first molecular evidence for the contribution of an inhibitory component to glutamatergic signaling in S. mansoni. Our findings add to a growing body of evidence suggesting that glutamatergic signaling in schistosomes may be physiologically important, and could be targeted for chemotherapeutic intervention.