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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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Phylogenetic relationship between the SmGluCl subunits and the inhibitory Cys-loop LGIC family.Maximum likelihood tree showing the evolutionary relationship of S. mansoni GluCl subunits compared to other Cys-loop anionic LGIC subunits. A. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to an independent glutamate-gated chloride channel clade evolutionarily distinct from their mollusc, arthropod and nematode counterparts. No schistosome Cys-loop LGIC subunits are predicted to mediate GABA signaling. All other LGIC subunits predicted from S. mansoni genome belong to the nAChR cation channel family (not shown). The phylogenetic analysis included inhibitory Cys-loop LGIC subunits from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Haemonchus contortus (Hco), the snail species Aplysia californica (Ac), Haliotis asinina (Ha) and Lymnaea stagnalis (Ls), and Rattus norvegicus (Rn). The cation-selective GABA-gated channel subunits CeEXP-1, CeLGC-35, DmGRD and DmLCCH3 were included as part of the inhibitory Cys-loop LGIC family. The tree was rooted using C. elegans nAChR cation channel subunits ACR-11, DEG-3, UNC-29 and UNC-38 as outlier. B. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to the flatworm GluCl subunit clade that includes other trematode and cestode putative GluCl-like subunits. The flatworm GluCl family is evolutionarily distinct from the mollusc, arthropod and nematode GluCl families. The phylogenetic analysis included the GluCl and GlyR subunits from panel A, as well as GluCl-like subunits from the trematode S. japonicum (Sjp), S. haematobium (Sha) and C. sinensis (Csi), and the cestodes E. multilocularis (EmuJ) and H. microstoma (HmN). For both trees, subunits for which the function had not been confirmed by heterologous expression are labeled in gray. S. mansoni GluCl subunits are in bold. Amino acid sequences were aligned with PROMALS3D and non-alignable, non-informative sites were removed manually. The Phylogeny.fr platform was used for tree building (PhyML v3.0, WAG substitution model). Numbers on internal branches indicate reliability (%) for internal branches and were assessed using the aLRT test (Chi2-based parametric).
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ppat-1003586-g006: Phylogenetic relationship between the SmGluCl subunits and the inhibitory Cys-loop LGIC family.Maximum likelihood tree showing the evolutionary relationship of S. mansoni GluCl subunits compared to other Cys-loop anionic LGIC subunits. A. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to an independent glutamate-gated chloride channel clade evolutionarily distinct from their mollusc, arthropod and nematode counterparts. No schistosome Cys-loop LGIC subunits are predicted to mediate GABA signaling. All other LGIC subunits predicted from S. mansoni genome belong to the nAChR cation channel family (not shown). The phylogenetic analysis included inhibitory Cys-loop LGIC subunits from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Haemonchus contortus (Hco), the snail species Aplysia californica (Ac), Haliotis asinina (Ha) and Lymnaea stagnalis (Ls), and Rattus norvegicus (Rn). The cation-selective GABA-gated channel subunits CeEXP-1, CeLGC-35, DmGRD and DmLCCH3 were included as part of the inhibitory Cys-loop LGIC family. The tree was rooted using C. elegans nAChR cation channel subunits ACR-11, DEG-3, UNC-29 and UNC-38 as outlier. B. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to the flatworm GluCl subunit clade that includes other trematode and cestode putative GluCl-like subunits. The flatworm GluCl family is evolutionarily distinct from the mollusc, arthropod and nematode GluCl families. The phylogenetic analysis included the GluCl and GlyR subunits from panel A, as well as GluCl-like subunits from the trematode S. japonicum (Sjp), S. haematobium (Sha) and C. sinensis (Csi), and the cestodes E. multilocularis (EmuJ) and H. microstoma (HmN). For both trees, subunits for which the function had not been confirmed by heterologous expression are labeled in gray. S. mansoni GluCl subunits are in bold. Amino acid sequences were aligned with PROMALS3D and non-alignable, non-informative sites were removed manually. The Phylogeny.fr platform was used for tree building (PhyML v3.0, WAG substitution model). Numbers on internal branches indicate reliability (%) for internal branches and were assessed using the aLRT test (Chi2-based parametric).

Mentions: Seventeen Cys-loop LGIC subunits are predicted in the S. mansoni genome: the four inhibitory subunits described here and 13 other putative nAChR-like Cys-loop LGIC subunits [14]. We first examined the phylogenetic relationship between the four inhibitory Cys-loop LGIC subunits cloned from S. mansoni and representatives of vertebrate and invertebrate inhibitory and excitatory Cys-loop LGIC subunits (Figure 6A). The phylogenetic analysis included inhibitory subunits from C. elegans, H. contortus, D. melanogaster, the snail species A. californica, Haliotis asinina and Lymnaea stagnalis, and Rattus norvegicus. Several subunits homologous to the SmGluCls were identified by protein BLAST search against the genomes of related trematode and cestode species: S. haematobium[40], [72], S. japonicum[73], C. sinensis[41], E. multilocularis and H. microstoma[42]. We examined the phylogenetic relationship between the putative inhibitory Cys-loop LGIC subunits identified in these flatworms, the inhibitory Cys-loop LGIC subunits cloned from S. mansoni, and GluCl subunits from the snail, nematode and insect species mentioned above (Figure 6B).


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)

Phylogenetic relationship between the SmGluCl subunits and the inhibitory Cys-loop LGIC family.Maximum likelihood tree showing the evolutionary relationship of S. mansoni GluCl subunits compared to other Cys-loop anionic LGIC subunits. A. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to an independent glutamate-gated chloride channel clade evolutionarily distinct from their mollusc, arthropod and nematode counterparts. No schistosome Cys-loop LGIC subunits are predicted to mediate GABA signaling. All other LGIC subunits predicted from S. mansoni genome belong to the nAChR cation channel family (not shown). The phylogenetic analysis included inhibitory Cys-loop LGIC subunits from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Haemonchus contortus (Hco), the snail species Aplysia californica (Ac), Haliotis asinina (Ha) and Lymnaea stagnalis (Ls), and Rattus norvegicus (Rn). The cation-selective GABA-gated channel subunits CeEXP-1, CeLGC-35, DmGRD and DmLCCH3 were included as part of the inhibitory Cys-loop LGIC family. The tree was rooted using C. elegans nAChR cation channel subunits ACR-11, DEG-3, UNC-29 and UNC-38 as outlier. B. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to the flatworm GluCl subunit clade that includes other trematode and cestode putative GluCl-like subunits. The flatworm GluCl family is evolutionarily distinct from the mollusc, arthropod and nematode GluCl families. The phylogenetic analysis included the GluCl and GlyR subunits from panel A, as well as GluCl-like subunits from the trematode S. japonicum (Sjp), S. haematobium (Sha) and C. sinensis (Csi), and the cestodes E. multilocularis (EmuJ) and H. microstoma (HmN). For both trees, subunits for which the function had not been confirmed by heterologous expression are labeled in gray. S. mansoni GluCl subunits are in bold. Amino acid sequences were aligned with PROMALS3D and non-alignable, non-informative sites were removed manually. The Phylogeny.fr platform was used for tree building (PhyML v3.0, WAG substitution model). Numbers on internal branches indicate reliability (%) for internal branches and were assessed using the aLRT test (Chi2-based parametric).
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Related In: Results  -  Collection

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

ppat-1003586-g006: Phylogenetic relationship between the SmGluCl subunits and the inhibitory Cys-loop LGIC family.Maximum likelihood tree showing the evolutionary relationship of S. mansoni GluCl subunits compared to other Cys-loop anionic LGIC subunits. A. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to an independent glutamate-gated chloride channel clade evolutionarily distinct from their mollusc, arthropod and nematode counterparts. No schistosome Cys-loop LGIC subunits are predicted to mediate GABA signaling. All other LGIC subunits predicted from S. mansoni genome belong to the nAChR cation channel family (not shown). The phylogenetic analysis included inhibitory Cys-loop LGIC subunits from Caenorhabditis elegans (Ce), Drosophila melanogaster (Dm), Haemonchus contortus (Hco), the snail species Aplysia californica (Ac), Haliotis asinina (Ha) and Lymnaea stagnalis (Ls), and Rattus norvegicus (Rn). The cation-selective GABA-gated channel subunits CeEXP-1, CeLGC-35, DmGRD and DmLCCH3 were included as part of the inhibitory Cys-loop LGIC family. The tree was rooted using C. elegans nAChR cation channel subunits ACR-11, DEG-3, UNC-29 and UNC-38 as outlier. B. SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits belong to the flatworm GluCl subunit clade that includes other trematode and cestode putative GluCl-like subunits. The flatworm GluCl family is evolutionarily distinct from the mollusc, arthropod and nematode GluCl families. The phylogenetic analysis included the GluCl and GlyR subunits from panel A, as well as GluCl-like subunits from the trematode S. japonicum (Sjp), S. haematobium (Sha) and C. sinensis (Csi), and the cestodes E. multilocularis (EmuJ) and H. microstoma (HmN). For both trees, subunits for which the function had not been confirmed by heterologous expression are labeled in gray. S. mansoni GluCl subunits are in bold. Amino acid sequences were aligned with PROMALS3D and non-alignable, non-informative sites were removed manually. The Phylogeny.fr platform was used for tree building (PhyML v3.0, WAG substitution model). Numbers on internal branches indicate reliability (%) for internal branches and were assessed using the aLRT test (Chi2-based parametric).
Mentions: Seventeen Cys-loop LGIC subunits are predicted in the S. mansoni genome: the four inhibitory subunits described here and 13 other putative nAChR-like Cys-loop LGIC subunits [14]. We first examined the phylogenetic relationship between the four inhibitory Cys-loop LGIC subunits cloned from S. mansoni and representatives of vertebrate and invertebrate inhibitory and excitatory Cys-loop LGIC subunits (Figure 6A). The phylogenetic analysis included inhibitory subunits from C. elegans, H. contortus, D. melanogaster, the snail species A. californica, Haliotis asinina and Lymnaea stagnalis, and Rattus norvegicus. Several subunits homologous to the SmGluCls were identified by protein BLAST search against the genomes of related trematode and cestode species: S. haematobium[40], [72], S. japonicum[73], C. sinensis[41], E. multilocularis and H. microstoma[42]. We examined the phylogenetic relationship between the putative inhibitory Cys-loop LGIC subunits identified in these flatworms, the inhibitory Cys-loop LGIC subunits cloned from S. mansoni, and GluCl subunits from the snail, nematode and insect species mentioned above (Figure 6B).

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