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Molecular cloning and characterization of novel glutamate-gated chloride channel subunits from Schistosoma mansoni.

Dufour V, Beech RN, Wever C, Dent JA, Geary TG - PLoS Pathog. (2013)

Bottom Line: We found no evidence of GABA receptors in S. mansoni.SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Host-Parasite Interactions, Institute of Parasitology, McGill University-MacDonald Campus, Sainte-Anne-de-Bellevue, Québec, Canada.

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.

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L-glutamate concentration-response relationships of SmGluCl-1:SmGluCl-2.1 and SmGluCl-1:SmGluCl-3 hetero-oligomers in Xenopus oocytes.L-glutamate concentration-response relationships from oocytes injected with a 1∶1 ratio of SmGluCl-1 cRNA and SmGluCl-2.1 or SmGluCl-3 cRNA. A. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-2.1 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-2.1cRNAs. Co-injection of SmGluCl-1 cRNA with SmGluCl-2.1 cRNA decreased the response to glutamate compared to oocytes injected with SmGluCl-2.1 cRNA alone (P<0.05, paired-T test), suggesting that SmGluCl-1 is a GluCl subunit and can form a heteromeric receptor with SmGluCl-2.1. B. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-3 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-3 cRNAs. Co-injection of SmGluCl-1 and SmGluCl-3 cRNAs did not alter the concentration-response relationship to glutamate compared to SmGluCl-3 homomeric receptors (P>0.05, paired-T test), suggesting that SmGluCl-1 cannot form a hetero-oligomer with SmGluCl-3 in Xenopus oocytes. All experiments were performed at a holding potential of −80 mV. For the concentration-response curves, responses to each application were normalized by assigning 100% to the maximum amplitude of the response to L-glutamate. N = 3 (where N is batches of oocytes) and n = 8 (where n is the number of individual oocytes) for each data point. Values of EC50 and the Hill coefficient are indicated as the mean ± SE. Error bars represent SD.
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ppat-1003586-g004: L-glutamate concentration-response relationships of SmGluCl-1:SmGluCl-2.1 and SmGluCl-1:SmGluCl-3 hetero-oligomers in Xenopus oocytes.L-glutamate concentration-response relationships from oocytes injected with a 1∶1 ratio of SmGluCl-1 cRNA and SmGluCl-2.1 or SmGluCl-3 cRNA. A. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-2.1 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-2.1cRNAs. Co-injection of SmGluCl-1 cRNA with SmGluCl-2.1 cRNA decreased the response to glutamate compared to oocytes injected with SmGluCl-2.1 cRNA alone (P<0.05, paired-T test), suggesting that SmGluCl-1 is a GluCl subunit and can form a heteromeric receptor with SmGluCl-2.1. B. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-3 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-3 cRNAs. Co-injection of SmGluCl-1 and SmGluCl-3 cRNAs did not alter the concentration-response relationship to glutamate compared to SmGluCl-3 homomeric receptors (P>0.05, paired-T test), suggesting that SmGluCl-1 cannot form a hetero-oligomer with SmGluCl-3 in Xenopus oocytes. All experiments were performed at a holding potential of −80 mV. For the concentration-response curves, responses to each application were normalized by assigning 100% to the maximum amplitude of the response to L-glutamate. N = 3 (where N is batches of oocytes) and n = 8 (where n is the number of individual oocytes) for each data point. Values of EC50 and the Hill coefficient are indicated as the mean ± SE. Error bars represent SD.

Mentions: To assess whether the SmGluCl-1 subunit could form a functional glutamate receptor, we co-expressed it with the other subunits. The underlying rationale was that, since a homomeric SmGluCl-1 receptor is not detectable in Xenopus oocytes, any shift in the concentration-response to L-glutamate relative to the homomeric receptors would be indicative of functional heteromeric glutamate receptors containing SmGluCl-1 subunits. We determined the L-glutamate concentration-response relationships of oocytes injected with a 1∶1 ratio of SmGluCl-1 cRNA and either SmGluCl-2.1 cRNA (SmGluCl-1:SmGluCl-2.1), or SmGluCl-3 cRNA (SmGluCl-1:SmGluCl-3). Co-injection of SmGluCl-1 and SmGluCl-2.1 cRNAs decreased sensitivity to L-glutamate compared to oocytes injected with SmGluCl-2.1 cRNA alone (P<0.05, paired-T test), confirming that SmGluCl-1 is a glutamate receptor subunit able to form heteromeric receptors with SmGluCl-2.1. The EC50 for L-glutamate on heteromeric SmGluCl-1:SmGluCl-2.1 receptors increased to 26.3±2.0 µM, while the Hill coefficient decreased to 1.2±0.1 (n = 8, Figure 4A), indicating significant loss of cooperativity in the heteromeric receptors compared to homomeric SmGluCl-2.1 receptors. It should be noted that the co-injection of a 1∶1 ratio of SmGluCl-1 and SmGluCl-2.1 cRNAs likely produced more than one population of heteromeric receptors with distinct subunit stoichiometries, along with a third population of homomeric SmGluCl-2.1 receptors. The concentration-response obtained reflects the contribution of the different populations of receptors present. It is not known whether the SmGluCl-1 and SmGluCl-2 combination occurs naturally in S. mansoni. In contrast, the evidence did not support the formation of heteromeric SmGluCl-1:SmGluCl-3 receptors. The EC50 and Hill coefficient for L-glutamate on SmGluCl-1:SmGluCl-3-injected oocytes were 7.0±0.7 µM and 1.0±0.1, respectively (n = 8, Figure 4B), indicating that co-injection of SmGluCl-1:SmGluCl-3 cRNAs did not alter sensitivity to L-glutamate compared to homomeric SmGluCl-3 receptors (P>0.05, paired-T test).


Molecular cloning and characterization of novel glutamate-gated chloride channel subunits from Schistosoma mansoni.

Dufour V, Beech RN, Wever C, Dent JA, Geary TG - PLoS Pathog. (2013)

L-glutamate concentration-response relationships of SmGluCl-1:SmGluCl-2.1 and SmGluCl-1:SmGluCl-3 hetero-oligomers in Xenopus oocytes.L-glutamate concentration-response relationships from oocytes injected with a 1∶1 ratio of SmGluCl-1 cRNA and SmGluCl-2.1 or SmGluCl-3 cRNA. A. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-2.1 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-2.1cRNAs. Co-injection of SmGluCl-1 cRNA with SmGluCl-2.1 cRNA decreased the response to glutamate compared to oocytes injected with SmGluCl-2.1 cRNA alone (P<0.05, paired-T test), suggesting that SmGluCl-1 is a GluCl subunit and can form a heteromeric receptor with SmGluCl-2.1. B. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-3 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-3 cRNAs. Co-injection of SmGluCl-1 and SmGluCl-3 cRNAs did not alter the concentration-response relationship to glutamate compared to SmGluCl-3 homomeric receptors (P>0.05, paired-T test), suggesting that SmGluCl-1 cannot form a hetero-oligomer with SmGluCl-3 in Xenopus oocytes. All experiments were performed at a holding potential of −80 mV. For the concentration-response curves, responses to each application were normalized by assigning 100% to the maximum amplitude of the response to L-glutamate. N = 3 (where N is batches of oocytes) and n = 8 (where n is the number of individual oocytes) for each data point. Values of EC50 and the Hill coefficient are indicated as the mean ± SE. Error bars represent SD.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757052&req=5

ppat-1003586-g004: L-glutamate concentration-response relationships of SmGluCl-1:SmGluCl-2.1 and SmGluCl-1:SmGluCl-3 hetero-oligomers in Xenopus oocytes.L-glutamate concentration-response relationships from oocytes injected with a 1∶1 ratio of SmGluCl-1 cRNA and SmGluCl-2.1 or SmGluCl-3 cRNA. A. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-2.1 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-2.1cRNAs. Co-injection of SmGluCl-1 cRNA with SmGluCl-2.1 cRNA decreased the response to glutamate compared to oocytes injected with SmGluCl-2.1 cRNA alone (P<0.05, paired-T test), suggesting that SmGluCl-1 is a GluCl subunit and can form a heteromeric receptor with SmGluCl-2.1. B. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-3 cRNA alone or injected with a 1∶1 ratio of SmGluCl-1 and SmGluCl-3 cRNAs. Co-injection of SmGluCl-1 and SmGluCl-3 cRNAs did not alter the concentration-response relationship to glutamate compared to SmGluCl-3 homomeric receptors (P>0.05, paired-T test), suggesting that SmGluCl-1 cannot form a hetero-oligomer with SmGluCl-3 in Xenopus oocytes. All experiments were performed at a holding potential of −80 mV. For the concentration-response curves, responses to each application were normalized by assigning 100% to the maximum amplitude of the response to L-glutamate. N = 3 (where N is batches of oocytes) and n = 8 (where n is the number of individual oocytes) for each data point. Values of EC50 and the Hill coefficient are indicated as the mean ± SE. Error bars represent SD.
Mentions: To assess whether the SmGluCl-1 subunit could form a functional glutamate receptor, we co-expressed it with the other subunits. The underlying rationale was that, since a homomeric SmGluCl-1 receptor is not detectable in Xenopus oocytes, any shift in the concentration-response to L-glutamate relative to the homomeric receptors would be indicative of functional heteromeric glutamate receptors containing SmGluCl-1 subunits. We determined the L-glutamate concentration-response relationships of oocytes injected with a 1∶1 ratio of SmGluCl-1 cRNA and either SmGluCl-2.1 cRNA (SmGluCl-1:SmGluCl-2.1), or SmGluCl-3 cRNA (SmGluCl-1:SmGluCl-3). Co-injection of SmGluCl-1 and SmGluCl-2.1 cRNAs decreased sensitivity to L-glutamate compared to oocytes injected with SmGluCl-2.1 cRNA alone (P<0.05, paired-T test), confirming that SmGluCl-1 is a glutamate receptor subunit able to form heteromeric receptors with SmGluCl-2.1. The EC50 for L-glutamate on heteromeric SmGluCl-1:SmGluCl-2.1 receptors increased to 26.3±2.0 µM, while the Hill coefficient decreased to 1.2±0.1 (n = 8, Figure 4A), indicating significant loss of cooperativity in the heteromeric receptors compared to homomeric SmGluCl-2.1 receptors. It should be noted that the co-injection of a 1∶1 ratio of SmGluCl-1 and SmGluCl-2.1 cRNAs likely produced more than one population of heteromeric receptors with distinct subunit stoichiometries, along with a third population of homomeric SmGluCl-2.1 receptors. The concentration-response obtained reflects the contribution of the different populations of receptors present. It is not known whether the SmGluCl-1 and SmGluCl-2 combination occurs naturally in S. mansoni. In contrast, the evidence did not support the formation of heteromeric SmGluCl-1:SmGluCl-3 receptors. The EC50 and Hill coefficient for L-glutamate on SmGluCl-1:SmGluCl-3-injected oocytes were 7.0±0.7 µM and 1.0±0.1, respectively (n = 8, Figure 4B), indicating that co-injection of SmGluCl-1:SmGluCl-3 cRNAs did not alter sensitivity to L-glutamate compared to homomeric SmGluCl-3 receptors (P>0.05, paired-T test).

Bottom Line: We found no evidence of GABA receptors in S. mansoni.SmGluCl receptors are insensitive to 1 µM ivermectin (IVM), indicating that they do not belong to the highly IVM-sensitive GluClα subtype group.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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Host-Parasite Interactions, Institute of Parasitology, McGill University-MacDonald Campus, Sainte-Anne-de-Bellevue, Québec, Canada.

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.

Show MeSH
Related in: MedlinePlus