Figure 1.
Predicted ion-selectivity of putative S. mansoni nAChRs.
A structural alignment of human, Lymnaea and S. mansoni nAChR subunits was generated using the Torpedo nAChR structure (PDB Accession # 2BG9) as a template. The M1-M2 linker region, shown here, is a key determinant of ion-selectivity in Cys-loop ligand gated ion channels. A glutamate residue (arrow) confers cation-selectivity and is present in all vertebrate subunits, as well as two of the S. mansoni subunits. The remaining schistosome and snail subunits display a Pro-Ala motif in this position, suggesting anion-selectivity. The two subunits described in this study are identified as S. mansoni acetylcholine-gated chloride channels SmACC-1 and SmACC-2. Other S. mansoni subunits are identified by their “Smp” designation obtained from the S. mansoni Genome Database (S. mansoni GeneDB). The corresponding GenBank accession numbers are listed in Table S1.
Figure 2.
Phylogenetic analysis of cys-loop ion channel subunits.
A bootstrapped, neighbor-joining tree was constructed in PHYLIP from a CLUSTALX alignment of vertebrate and invertebrate Cys-loop superfamily receptor subunits. The tree is midpoint-rooted and was visualized using FigTree 3.0. Only nodes supported by bootstrap values of 70% or higher are shown. Two distinct groups of receptors can be seen, the γ-aminobutyric acid (GABA)/glycine-like anion channels and the nicotinic acetylcholine receptors (nAChRs). The C. elegans acetylcholine-gated chloride channels (ACC) form a distinct clade within the larger group of GABA/glycine anion channels (green inset). In contrast the predicted Schistosoma acetylcholine-gated chloride channels (SmACCs) align with cholinergic nicotinic nAChRs, suggesting divergent evolutionary paths. The SmACCs described here are indicated by arrows and they constitute a separate clade in the nAChR tree along with putative homologs from flatworms Dugesia (Dtig), Clonorchis (Cs) and S. haematobium, as well as the snail Lymnaea (Lym). Accession numbers for sequences used in the alignment are listed in Table S1.
Figure 3.
Pharmacological and RNAi behavioral assays in schistosomula.
(A) Relative motility of 6-day old larvae was measured before and 5 minutes after the addition of cholinergic compounds, each at 100 µM. Data were normalized to baseline motility measured before drug addition. The data are the means and SEM of three independent experiments, each containing at least 12 animals. (B) Freshly transformed schistosomula were transfected with 50 nM irrelevant (scrambled) siRNA or 50 nM siRNA targeting a specific subunit. The following subunits were targeted in this study (refer to Table S1 for Accession numbers): Smp_157, Smp_157790; Smp_132, Smp_132070; Smp_037, Smp_037960; SmACC-1, Smp_176310; SmACC-2, Smp_142690. Larval motility was measured 6 days post-transfection and normalized relative to untransfected larvae cultured for the same period of time. The data shown are the means of three independent experiments, each containing at least 12 animals. *Significantly different from the scrambled siRNA control at P<0.05.
Figure 4.
Confirmation of SmACC knockdown.
(A) Knockdown of SmACC-1 and SmACC-2 was confirmed at the mRNA level. Schistosomula were transfected with subunit-specific siRNA or scrambled siRNA control, as indicated. RNA was extracted at 6 days post-transfection, oligo-dT reverse-transcribed and quantitative qPCR was performed using primers targeting either a specific subunit or a non-relevant SmACC subunit as an off-target control. Relative expression was calculated using the comparative ΔΔCt method after normalization to a housekeeping gene (GAPDH). The data are derived from three independent experiments, each with 3 replicates, and are shown as the % remaining expression relative to the scrambled siRNA control. Silencing of both subunits was statistically significant, as measured by a Student's t-test at P<0.05 (B) Western blot analysis was performed to assay for silencing of SmACC-1 at the protein level. Crude membrane protein extracts from SmACC-1 siRNA-treated and negative control schistosomula were resolved on a SDS-PAGE gel, transferred to a PVDF membrane and probed with affinity-purified anti-SmACC-1 or a loading control (anti-Sm5-HTR [74]). A band of the expected size (arrow) is present in the scrambled negative control lane but not in the SmACC-1 siRNA-treated lane.
Figure 5.
Immunolocalization of SmACC-1 and SmACC-2 in Schistosoma mansoni.
Adult and 6-day old schistosomula were fixed and incubated with affinity-purified anti-SmACC-1 or anti-SmACC-2, followed by Alexa 488-conjugated secondary antibody (green). In some animals the body wall musculature was counterstained with tetramethylrhodamine B isothiocyanate (TRITC)-labeled phalloidin (red). (A) A Z-projection of SmACC-1 immunoreactivity in an adult male worm. SmACC-1 is present in both the oral sucker (os) and in minor nerve fibers of the peripheral innervation of the worm's body wall. The nerve fibers are varicose in appearance, resembling beads on a string (enlarged region, solid arrows) and are repeated along the length of the body. The asterisk (*) indicates an area of non-specific fluorescence resulting from tissue damage (B) Z-projection of an adult male worm labeled with anti-SmACC-2 (green) and phalloidin (red). SmACC-2 immunoreactivity is present in varicose nerve fibers (solid arrows) that cross the body in a mesh-like pattern indicative of PNS staining. SmACC-2 and the phalloidin –stained body wall musculature are present at different depths of the animal, suggesting that SmACC-2 does not directly innervate muscle. (C) Tubercles (tb) of an adult male worm labeled with anti-SmACC-2 and phalloidin. Specific, punctate SmACC-2 immunoreactivity can be seen along the surface and within the tubercles (arrows). (D) SmACC-2 forms a pattern of concentric, varicose nerve fibers that run the entire length of a 6-day old schistosomulum. A similar expression pattern was observed in schistosomula labeled with anti-SmACC-1 antibody (not shown). (E) Transmitted light and corresponding fluorescent image of a negative control worm labeled with peptide-preadsorbed anti-SmACC-1 and (F) the same negative control for peptide-preadsorbed anti-SmACC-2. The scale bars for the two negative controls are 50 µm (panel E) and 20 µm (panel F).
Figure 6.
Functional characterization of SmACC-1 in HEK-293 cells.
HEK-293 cells were transfected with a human codon-optimized SmACC-1 construct and labeled with affinity-purified anti-SmACC-1 antibody, followed by FITC-conjugated secondary antibody (green). (A) The results show specific immunoreactivity along the surface of the cells, consistent with protein expression. (B) No immunofluorescence is present in cells transfected with empty vector (mock control). (C) Schematic representation of the Premo Halide Sensor YFP quench assay. Cells expressing YFP and the chloride channel of interest are bathed in buffer containing iodide (I−), which is used as a surrogate for chloride ions. Agonist-induced activation of the channel causes an influx of I− into the cell and quenches YFP fluorescence. (D) Representative data from individual wells containing cells transfected with either SmACC-1 or empty vector (mock). Treatment of SmACC-1 expressing cells with 100 µM nicotine (solid red squares) resulted in a significant reduction in YFP fluorescence (YFP quench) when compared to both a water-treated negative control (solid triangles) and mock-transfected cells treated with 100 µM nicotine (solid circles). Data were normalized relative to maximum YFP fluorescence for each sample.
Figure 7.
SmACC-1 is selectively activated by cholinergic substances in transfected HEK-293 cells.
(A) A panel of cholinergic receptor agonists (acetylcholine (ACh), choline, carbachol, nicotine, arecoline) was tested against SmACC-1 expressing or mock-transfected cells. The YFP quench data were normalized relative to the water-treated control measured in the same experiment and on the same plate. Results are the means and SEM of 3-4 experiments, each containing 6 technical replicates per treatment. All cholinergic agonists caused a significant reduction in YFP fluorescence at P<0.05 (*) compared to the water control. Treatment of SmACC-1-expressing cells with serotonin (5HT), glutamate or dopamine did not result in significant YFP quench. (B) SmACC-1 expressing cells were treated with variable concentrations of nicotine and YFP quench was calculated. The YFP quench data were normalized relative to the maximum response for each experiment and an EC50 value was calculated by nonlinear regression analysis of the normalized data. The results are the means ± SEM of 3 independent experiments, each with six replicates.
Figure 8.
SmACC-1 is selectively antagonized by D-tubocurarine in transfected HEK-293 cells.
SmACC-1-expressing cells were pre-incubated with cholinergic antagonists (mecamylamine, D-tubocurarine, atropine), each at a concentration of 100 µM. Cells were then treated with 100 µM nicotine and YFP quench was measured. Control SmACC-1 cells were treated with nicotine in the absence of antagonist. The YFP quench data were normalized relative to the water-treated control. Results are the means and SEM of 12 replicates from two separate transfections.