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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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Ion selectivity of SmGluCl-2.1 and SmGluCl-3.Glutamate-sensitive current-voltage relationship experiments were performed in Xenopus oocytes injected with SmGluCl-2.1 or SmGluCl-3 cRNA. The M2 domain of all 4 members of the SmGluCl clade contains the molecular determinants for chloride selectivity, as shown in Figure S1[23], [24]. A. Current-voltage curves obtained from oocytes injected with SmGluCl-2.1 cRNA. A positive shift in the reversal potential is observed when the extracellular chloride concentration was altered (P<0.0001, one-way ANOVA), consistent with a chloride-selective SmGluCl-2.1 receptor. Extracellular chloride was 103.6 mM for normal ND96 and 45.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 38 mM for reduced sodium ND96. B. Current-voltage curves obtained from oocytes injected with SmGluCl-3 cRNA. A positive shift in the reversal potential was observed when the extracellular chloride concentration is altered (P<0.0001, one-way ANOVA), indicating that the SmGluCl-3 receptor exhibits chloride selectivity. Extracellular chloride was 103.6 mM for normal ND96 and 13.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 6 mM for reduced sodium ND96. Chloride or sodium were replaced with sodium gluconate or choline chloride, respectively. n = 4 (where n is the number of individual oocytes) for each data point. Error bars represent SD.
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ppat-1003586-g005: Ion selectivity of SmGluCl-2.1 and SmGluCl-3.Glutamate-sensitive current-voltage relationship experiments were performed in Xenopus oocytes injected with SmGluCl-2.1 or SmGluCl-3 cRNA. The M2 domain of all 4 members of the SmGluCl clade contains the molecular determinants for chloride selectivity, as shown in Figure S1[23], [24]. A. Current-voltage curves obtained from oocytes injected with SmGluCl-2.1 cRNA. A positive shift in the reversal potential is observed when the extracellular chloride concentration was altered (P<0.0001, one-way ANOVA), consistent with a chloride-selective SmGluCl-2.1 receptor. Extracellular chloride was 103.6 mM for normal ND96 and 45.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 38 mM for reduced sodium ND96. B. Current-voltage curves obtained from oocytes injected with SmGluCl-3 cRNA. A positive shift in the reversal potential was observed when the extracellular chloride concentration is altered (P<0.0001, one-way ANOVA), indicating that the SmGluCl-3 receptor exhibits chloride selectivity. Extracellular chloride was 103.6 mM for normal ND96 and 13.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 6 mM for reduced sodium ND96. Chloride or sodium were replaced with sodium gluconate or choline chloride, respectively. n = 4 (where n is the number of individual oocytes) for each data point. Error bars represent SD.

Mentions: In anionic LGICs, −2′ proline, −1′ alanine and 13′ threonine residues from the M2 transmembrane domain are the minimal determinants of anion selectivity [23], [24]. These three residues are conserved in the SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits (Figure S1), which are therefore predicted to be permeable to Cl−. To confirm that the SmGluCls form Cl− channels, we performed current-voltage (I/V) relationship experiments on oocytes expressing homomeric SmGluCl-2.1 and SmGluCl-3 receptors. For SmGluCl-2.1, we compared the reversal potentials for glutamate-sensitive currents in normal ND96 solution containing 103.6 mM Cl− and 96 mM Na+, sodium gluconate-replaced ND96 with [Cl−] reduced to 45.6 mM, and choline chloride-replaced ND96 with [Na+] reduced to 38 mM. In oocytes injected with SmGluCl-2.1 cRNA, reduction of external [Cl−] shifted the reversal potential by 15.5±2.9 mV (n = 4), from −23.0±2.3 mV to −8.2±2.4 mV (P<0.0001, one-way ANOVA), reasonably close to the 20.7 mV shift predicted by the Nernst equation (Figure 5A), indicating that SmGluCl-2.1 receptors are Cl− selective. In contrast, reducing the extracellular [Na+] did not alter the reversal potential (−22.0±3.0 mV, n = 4) or the I/V relationship compared to normal ND96, indicating that SmGluCl-2.1 receptors are not permeable to Na+. We measured the reversal potentials of SmGluCl-3 receptors for glutamate-sensitive currents in normal ND96, sodium gluconate-replaced ND96 with [Cl−] reduced to 13.6 mM, and choline chloride-replaced ND96 containing 6 mM Na+. In oocytes injected with SmGluCl-3 cRNA, reduction of the external [Cl−] shifted the reversal potential by 37.4±2.8 mV (n = 4), from −15.6±9.8 mV to 21.9±9.2 mV (P<0.0001, one-way ANOVA), reasonably close to the 51.5 mV shift predicted by the Nernst equation (Figure 5B), indicating that SmGluCl-3 receptors are permeable to Cl−. Reducing the extracellular [Na+] had no influence on the I/V relationship or the reversal potential (−17.7±4.9 mV, n = 4) compared to normal ND96, indicating that SmGluCl-3 receptors are not permeable to Na+. Our electrophysiology data clearly demonstrate that SmGluCl-2.1 and SmGluCl-3 receptors are permeable to Cl−, as predicted from their M2 regions. These results can be extrapolated to all four S. mansoni GluCl subunits (SmGluCls), since they all possess the canonical molecular determinants for anion selectivity.


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)

Ion selectivity of SmGluCl-2.1 and SmGluCl-3.Glutamate-sensitive current-voltage relationship experiments were performed in Xenopus oocytes injected with SmGluCl-2.1 or SmGluCl-3 cRNA. The M2 domain of all 4 members of the SmGluCl clade contains the molecular determinants for chloride selectivity, as shown in Figure S1[23], [24]. A. Current-voltage curves obtained from oocytes injected with SmGluCl-2.1 cRNA. A positive shift in the reversal potential is observed when the extracellular chloride concentration was altered (P<0.0001, one-way ANOVA), consistent with a chloride-selective SmGluCl-2.1 receptor. Extracellular chloride was 103.6 mM for normal ND96 and 45.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 38 mM for reduced sodium ND96. B. Current-voltage curves obtained from oocytes injected with SmGluCl-3 cRNA. A positive shift in the reversal potential was observed when the extracellular chloride concentration is altered (P<0.0001, one-way ANOVA), indicating that the SmGluCl-3 receptor exhibits chloride selectivity. Extracellular chloride was 103.6 mM for normal ND96 and 13.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 6 mM for reduced sodium ND96. Chloride or sodium were replaced with sodium gluconate or choline chloride, respectively. n = 4 (where n is the number of individual oocytes) for each data point. Error bars represent SD.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003586-g005: Ion selectivity of SmGluCl-2.1 and SmGluCl-3.Glutamate-sensitive current-voltage relationship experiments were performed in Xenopus oocytes injected with SmGluCl-2.1 or SmGluCl-3 cRNA. The M2 domain of all 4 members of the SmGluCl clade contains the molecular determinants for chloride selectivity, as shown in Figure S1[23], [24]. A. Current-voltage curves obtained from oocytes injected with SmGluCl-2.1 cRNA. A positive shift in the reversal potential is observed when the extracellular chloride concentration was altered (P<0.0001, one-way ANOVA), consistent with a chloride-selective SmGluCl-2.1 receptor. Extracellular chloride was 103.6 mM for normal ND96 and 45.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 38 mM for reduced sodium ND96. B. Current-voltage curves obtained from oocytes injected with SmGluCl-3 cRNA. A positive shift in the reversal potential was observed when the extracellular chloride concentration is altered (P<0.0001, one-way ANOVA), indicating that the SmGluCl-3 receptor exhibits chloride selectivity. Extracellular chloride was 103.6 mM for normal ND96 and 13.6 mM for reduced chloride ND96. Extracellular sodium was 96 mM for normal ND96 and 6 mM for reduced sodium ND96. Chloride or sodium were replaced with sodium gluconate or choline chloride, respectively. n = 4 (where n is the number of individual oocytes) for each data point. Error bars represent SD.
Mentions: In anionic LGICs, −2′ proline, −1′ alanine and 13′ threonine residues from the M2 transmembrane domain are the minimal determinants of anion selectivity [23], [24]. These three residues are conserved in the SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4 subunits (Figure S1), which are therefore predicted to be permeable to Cl−. To confirm that the SmGluCls form Cl− channels, we performed current-voltage (I/V) relationship experiments on oocytes expressing homomeric SmGluCl-2.1 and SmGluCl-3 receptors. For SmGluCl-2.1, we compared the reversal potentials for glutamate-sensitive currents in normal ND96 solution containing 103.6 mM Cl− and 96 mM Na+, sodium gluconate-replaced ND96 with [Cl−] reduced to 45.6 mM, and choline chloride-replaced ND96 with [Na+] reduced to 38 mM. In oocytes injected with SmGluCl-2.1 cRNA, reduction of external [Cl−] shifted the reversal potential by 15.5±2.9 mV (n = 4), from −23.0±2.3 mV to −8.2±2.4 mV (P<0.0001, one-way ANOVA), reasonably close to the 20.7 mV shift predicted by the Nernst equation (Figure 5A), indicating that SmGluCl-2.1 receptors are Cl− selective. In contrast, reducing the extracellular [Na+] did not alter the reversal potential (−22.0±3.0 mV, n = 4) or the I/V relationship compared to normal ND96, indicating that SmGluCl-2.1 receptors are not permeable to Na+. We measured the reversal potentials of SmGluCl-3 receptors for glutamate-sensitive currents in normal ND96, sodium gluconate-replaced ND96 with [Cl−] reduced to 13.6 mM, and choline chloride-replaced ND96 containing 6 mM Na+. In oocytes injected with SmGluCl-3 cRNA, reduction of the external [Cl−] shifted the reversal potential by 37.4±2.8 mV (n = 4), from −15.6±9.8 mV to 21.9±9.2 mV (P<0.0001, one-way ANOVA), reasonably close to the 51.5 mV shift predicted by the Nernst equation (Figure 5B), indicating that SmGluCl-3 receptors are permeable to Cl−. Reducing the extracellular [Na+] had no influence on the I/V relationship or the reversal potential (−17.7±4.9 mV, n = 4) compared to normal ND96, indicating that SmGluCl-3 receptors are not permeable to Na+. Our electrophysiology data clearly demonstrate that SmGluCl-2.1 and SmGluCl-3 receptors are permeable to Cl−, as predicted from their M2 regions. These results can be extrapolated to all four S. mansoni GluCl subunits (SmGluCls), since they all possess the canonical molecular determinants for anion selectivity.

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