title: A high-throughput assay for modulators of ligand-gated chloride channels.
abstract: Invertebrate glutamate-gated chloride channels (GluCls) are important targets for anthelmintics and insecticides such as ivermectin. To facilitate screening for novel GluCl modulators, the Caenorhabditis elegans GluCl alpha2beta channel was chosen as a surrogate for parasite channels not yet cloned, and an inducible stable human embryonic kidney cell line was generated. Functional expression of the alpha2 and beta subunits was confirmed by whole-cell voltage clamp assays. Using this cell line, a high-throughput assay was developed that detects membrane potential changes associated with the activation of GluCls. In this assay, membrane depolarization was quantified via changes in fluorescence resonance energy transfer between two membrane-associated dyes. Robust and reproducible signals were detected in response to addition of glutamate or ivermectin. This assay was used for the screening of over 180,000 samples from natural and synthetic sources.
authors.affiliation: Merck Research Laboratories, Rahway, NJ 07065, USA. michel_hamelin@merck.com
authors.forename: Michel
authors.initials: M
authors.lastname: Hamelin
authors.forename: Xin
authors.initials: X
authors.lastname: Meng
authors.forename: Michael
authors.initials: M
authors.lastname: Cuddy
authors.forename: Keith
authors.initials: K
authors.lastname: Korsun
authors.forename: John
authors.initials: J
authors.lastname: Ondeyka
authors.forename: Peter B
authors.initials: PB
authors.lastname: Simpson
authors.forename: Doris F
authors.initials: DF
authors.lastname: Cully
authors.forename: Birgit T
authors.initials: BT
authors.lastname: Priest
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D017173:Caenorhabditis elegans; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000494:pharmacology*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012371:Robotics / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D018118:Chloride Channels; D011994:Recombinant Proteins; C109777:glutamate-gated chloride channels; D018698:Glutamic Acid