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 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.

Ion Channels and Drug Transporters as Targets for Anthelmintics

“…Schistosomes and other platyhelminths are typically not sensitive to macrocyclic lactones [53, 54] (though see [55]), which could suggest an absence of GluCl channels in these organisms. However, exciting recent work [56] has demonstrated that schistosomes in fact do express GluCl channel subunits, but that these subunits are phylogenetically distinct from those of other invertebrates, including nematodes, arthropods, and molluscs. When expressed in Xenopus oocytes, schistosome GluCl subunits form functional L-glutamate-gated, Cl − -permeable channels.…”

“…Macrocyclic lactones also appear to interact with nematode GABA-gated channels [57], and there is some evidence that the cyclooctadepsipeptide PF1022A (see below) binds to and interacts with nematode GABA receptors [59], though electrophysiological experiments suggest that it does not act as a GABA agonist [60]. Surprisingly, schistosomes do not appear to have genes for GABA-gated channels [56]. …”

The orphan pentameric ligand-gated ion channelpHCl-2is gated by pH and regulates fluid secretion inDrosophilaMalpighian tubules

“…Invertebrate genomes, in contrast, encode a greater assortment of channel types with a wider range of ligand specificities and ligand-ion combinations than those found in vertebrates (Dent, 2006). In addition to the nicotinic acetylcholine and GABA receptors found in vertebrates, invertebrate genomes encode anion-selective acetylcholine (Putrenko et al, 2005;van Nierop et al, 2005), histamine (Gengs et al, 2002;Zheng et al, 2002), glutamate (Dent et al, 1997;Dufour et al, 2013;Kehoe et al, 2009), serotonin (Ranganathan et al, 2000), dopamine (Ringstad et al, 2009), tyramine (Ringstad et al, 2009) and pH (Mounsey et al, 2007;Schnizler et al, 2005) channels, as well as cation-selective GABA (Gisselmann et al, 2004) and proton (Beg et al, 2008) channels. Moreover, multiple putative invertebrate pLGICs have been identified that cannot be assigned to any neurotransmitter family based on sequence homology.…”

A high-throughput colorimetric assay for detection of Schistosoma mansoni viability based on the tetrazolium salt XTT

“…These compounds were: the antimalarial drug mefloquine (MFQ) [27]; the antihelmintic Ivermectin (IVM) [28, 29]; the bromodomain inhibitor JQ1 [30]; and the reference drug praziquantel (PZQ). The positive and negative controls average absorbance values were used to calculate parasites viability.…”