Functional Characterization of a Novel Family of Acetylcholine-Gated Chloride Channels in Schistosoma mansoni

Abstract

Acetylcholine is the canonical excitatory neurotransmitter of the mammalian neuromuscular system. However, in the trematode parasite Schistosoma mansoni, cholinergic stimulation leads to muscle relaxation and a flaccid paralysis, suggesting an inhibitory mode of action. Information about the pharmacological mechanism of this inhibition is lacking. Here, we used a combination of techniques to assess the role of cholinergic receptors in schistosome motor function. The neuromuscular effects of acetylcholine are typically mediated by gated cation channels of the nicotinic receptor (nAChR) family. Bioinformatics analyses identified numerous nAChR subunits in the S. mansoni genome but, interestingly, nearly half of these subunits carried a motif normally associated with chloride-selectivity. These putative schistosome acetylcholine-gated chloride channels (SmACCs) are evolutionarily divergent from those of nematodes and form a unique clade within the larger family of nAChRs. Pharmacological and RNA interference (RNAi) behavioral screens were used to assess the role of the SmACCs in larval motor function. Treatment with antagonists produced the same effect as RNAi suppression of SmACCs; both led to a hypermotile phenotype consistent with abrogation of an inhibitory neuromuscular mediator. Antibodies were then generated against two of the SmACCs for use in immunolocalization studies. SmACC-1 and SmACC-2 localize to regions of the peripheral nervous system that innervate the body wall muscles, yet neither appears to be expressed directly on the musculature. One gene, SmACC-1, was expressed in HEK-293 cells and characterized using an iodide flux assay. The results indicate that SmACC-1 formed a functional homomeric chloride channel and was activated selectively by a panel of cholinergic agonists. The results described in this study identify a novel clade of nicotinic chloride channels that act as inhibitory modulators of schistosome neuromuscular function. Additionally, the iodide flux assay used to characterize SmACC-1 represents a new high-throughput tool for drug screening against these unique parasite ion channels.

A constitutively active G protein-coupled acetylcholine receptor regulates motility of larval Schistosoma mansoni

“…Due to their importance as antiparasitic drug targets, several nematode nicotinic receptors have been cloned and pharmacologically characterized [reviewed in 54]. Among the flatworms, putative cholinergic channel subunits have been cloned from S. haematobium [21] and we have recently described a first nicotinic chloride channel in S. mansoni [22]. …”

“…Given that the receptor is upregulated in early larval stages [10] and ACh is well known to control worm movement, we questioned whether SmGAR might play a role in the control of larval motility. RNAi is well established in schistosomes [60] and has been successful in elucidating the role of neuronal proteins in motor function [17, 22, 38]. Here, we modified a previously developed RNAi behavioral assay [17, 38] to assess the role of SmGAR in larval schistosomulae.…”

“…To explain the results it is important to keep in mind that SmGAR is one of many cholinergic receptors in schistosomes [7] and the flaccid paralysis caused by ACh in vitro [19, 20] is likely due to stimulation of multiple receptors all at once. Some of these receptors do have inhibitory effects on movement [22] but there could be stimulatory pathways that were missed in the earlier studies. Based on the RNAi data, SmGAR is the first example of a cholinergic receptor that stimulates schistosome motor activity, at least in the young larvae.…”

“…Schistosomes have several putative ACh receptors that may be responsible for this phenomenon [reviewed in 7]. The majority of these receptors are nicotinic ion channels, some of which have been cloned and characterized in vitro [21, 22]. However, two muscarinic cholinergic receptors are also predicted.…”

Ion Channels and Drug Transporters as Targets for Anthelmintics

“…Binding of extracellular acetylcholine opens the channel, allowing the flow of ions down the electrochemical gradient. nAchRs are typically cation selective and mediate excitatory responses by depolarizing the cell, though invertebrates also express anion-selective acetylcholine-gated channels that may play a role in fast inhibitory responses [25, 26]. As with other ligand-gated channels, acetylcholine also activates a separate class of metabotropic receptors that are not ion channels, and instead act through G-protein-coupled signaling pathways.…”