Dufour V et al. (2013), Molecular cloning and characterization of novel...

XB-ART-47924

PLoS Pathog 2013 Jan 01;98:e1003586. doi: 10.1371/journal.ppat.1003586.

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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 .

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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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Species referenced: Xenopus
Genes referenced: chrna4 ecd snai1

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	Figure 2. Functional expression of SmGluCl-1, SmGluCl-2, and SmGluCl-3 homo-oligomers in Xenopus oocytes.Two-electrode voltage-clamp experiments were performed in Xenopus oocytes injected with SmGluCl-1, SmGluCl-2.1 or SmGluCl-3 cRNA. Current traces evoked by 1 mM L-glutamate for SmGluCl-1, SmGluCl-2.1, and SmGluCl-3 homo-oligomers are shown. A–C. Oocytes injected with SmGluCl-1 cRNA alone produced no functional receptors, whereas oocytes injected with SmGluCl-2.1 cRNAs alone or SmGluCl-3 cRNA alone both produced functional homomeric receptors showing robust responses to application of L-glutamate. All experiments were performed at a holding potential of −80 mV. The period of agonist application is indicated by a bar above the trace. No agonists other than L-glutamate evoked any responses (not shown).
	Figure 3. L-glutamate concentration-response relationships of SmGluCl-2.1 and SmGluCl-3 homo-oligomers in Xenopus oocytes.A–B. Electrophysiological recording from an oocyte injected with SmGluCl-2 cRNA alone or SmGluCl-3 cRNA alone in response to the application of L-glutamate. The period of agonist application is indicated by a bar above the trace and the concentrations are given in µM. C. L-glutamate concentration-response relationships from oocytes injected with SmGluCl-2.1 cRNA alone or SmGluCl-3 cRNA alone. Oocytes injected with SmGluCl-3 cRNA exhibited greater sensitivity to L-glutamate than oocytes injected with SmGluCl-2 cRNA (P<0.05, paired-T test). All experiments were performed at a holding potential of −80 mV. For the concentration-response curves, responses to each application were normalized to the maximal 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.
	Figure 4. 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.
	Figure 5. 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.
	Figure 6. 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).
	Figure 1. Gene structure of SmGluCl-1, SmGluCl-2, SmGluCl-3 and SmGluCl-4.Gene structures of SmGluCl-1 (Smp_096480), SmGluCl-2 (Smp_015630), SmGluCl-3 (Smp_104890) and SmGluCl-4 (Smp_099500/Smp_176730).A–C. SmGluCl-1, SmGluCl-2 and SmGluCl-3 cloned cDNAs contain all the conserved domains and features characteristic of a full length, functional Cys-loop LGIC subunit. D. Full length SmGluCl-4 cDNA sequence could not be determined. SmGluCl-4 partially cloned cDNA lacks an initiation and termination codon, and does not encode a SP and M4. Contig misassembly could explain the 83,509 bp gap found between the two Cys-loop motifs of SmGluCl-4. Graphical representation of gene structures was performed with the Exon-Intron Graphic Maker version 4 (wormweb.org/exonintron). Scales are shown on the right side. Numbers next to the introns correspond to their size; the introns are not to scale. Smp_015630, Smp_096480, Smp_104890, Smp_099500 and Smp_176730 putative genes were obtained from the Schistosoma mansoni homepage on GeneDB (http://www.genedb.org/Homepage/Smansoni). SmGluCl-1 (1,536 bp; KC861381), SmGluCl-2.1 (1,659 bp; KC861382), SmGluCl-2.2 (1,638 bp; KC861383), SmGluCl-3 (1,545 bp, KC861384) and SmGluCl-4 (1,986 bp, KC861385) genes were cloned and amplified from adult worm cDNA. ECD, extracellular binding domain; ICD, intracellular domain; M1–4, membrane-spanning region 1–4; SP, signal peptide. Grey areas indicate regions that do not correspond to any known functional domains.

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