title: 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.
authors.affiliation: Institute of Parasitology, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec, Canada.
authors.forename: Kevin
authors.initials: K
authors.lastname: MacDonald
authors.affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Samuel
authors.initials: S
authors.lastname: Buxton
authors.affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kimber
authors.affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Tim A
authors.initials: TA
authors.lastname: Day
authors.affiliation: Department of Biomedical Sciences, Iowa State University, Ames, Iowa, United States of America.
authors.forename: Alan P
authors.initials: AP
authors.lastname: Robertson
authors.affiliation: Institute of Parasitology, McGill University, Macdonald Campus, Ste. Anne de Bellevue, Quebec, Canada.
authors.forename: Paula
authors.initials: P
authors.lastname: Ribeiro
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D000871:Anthelmintics / Q000627:therapeutic use; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D018679:Cholinergic Agonists / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D009043:Motor Activity / Q000187:drug effects* / Q000235:genetics; D018733:Nicotinic Antagonists / Q000494:pharmacology*; D011223:Praziquantel / Q000627:therapeutic use; D034622:RNA Interference; D034741:RNA, Small Interfering; D011950:Receptors, Cholinergic / Q000235:genetics; D011978:Receptors, Nicotinic / Q000235:genetics; D012550:Schistosoma mansoni / Q000235:genetics / Q000378:metabolism*; D012552:Schistosomiasis / Q000188:drug therapy / Q000473:pathology
chemical_list: D000871:Anthelmintics; D018118:Chloride Channels; D018679:Cholinergic Agonists; D018733:Nicotinic Antagonists; D034741:RNA, Small Interfering; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D011223:Praziquantel; D000109:Acetylcholine