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

K. MacDonald, S. Buxton, M.J. Kimber, T.A. Day, A.P. Robertson, P. Ribeiro

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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. © 2014 MacDonald et al.
Original languageEnglish
Article numbere1004181
Number of pages14
JournalPLoS Pathogens
Volume10
Issue number6
DOIs
Publication statusPublished - 12 Jun 2014

Bibliographical note

cited By 9

Keywords

  • acetylcholine
  • chloride channel
  • nicotinic receptor
  • small interfering RNA
  • anthelmintic agent
  • cholinergic receptor
  • cholinergic receptor stimulating agent
  • nicotinic receptor blocking agent
  • praziquantel
  • small interfering RNA, antibody production
  • article
  • bioinformatics
  • confocal microscopy
  • controlled study
  • gene expression
  • genetic transfection
  • genome analysis
  • immunolocalization
  • molecular cloning
  • motor performance
  • neuromuscular function
  • neurotransmitter release
  • nonhuman
  • parasite isolation
  • quantitative assay
  • reverse transcription polymerase chain reaction
  • RNA extraction
  • RNA interference
  • Schistosoma mansoni
  • schistosomiasis
  • Western blotting
  • animal
  • antagonists and inhibitors
  • cell line
  • drug effects
  • genetics
  • HEK293 cell line
  • human
  • metabolism
  • motor activity
  • pathology
  • schistosomiasis, Acetylcholine
  • Animals
  • Anthelmintics
  • Cell Line
  • Chloride Channels
  • Cholinergic Agonists
  • HEK293 Cells
  • Humans
  • Motor Activity
  • Nicotinic Antagonists
  • Praziquantel
  • Receptors, Cholinergic
  • Receptors, Nicotinic
  • RNA Interference
  • RNA, Small Interfering
  • Schistosomiasis

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  • Cite this

    MacDonald, K., Buxton, S., Kimber, M. J., Day, T. A., Robertson, A. P., & Ribeiro, P. (2014). Functional Characterization of a Novel Family of Acetylcholine-Gated Chloride Channels in Schistosoma mansoni. PLoS Pathogens, 10(6), [e1004181]. https://doi.org/10.1371/journal.ppat.1004181