title: Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay.
abstract: To develop a real-time thallium flux assay for high-throughput screening (HTS) of human KCNQ4 (Kv7.4) potassium channel openers, we used CHO-K1 cells stably expressing human KCNQ4 channel protein and a thallium-sensitive dye based on the permeability of thallium through potassium channels. The electrophysiological and pharmacological properties of the cell line expressing the KCNQ4 protein were found to be in agreement with that reported elsewhere. The EC(50) values of the positive control compound (retigabine) determined by the thallium and (86)rubidium flux assays were comparable to and consistent with those documented in the literature. Signal-to-background (S/B) ratio and Z factor of the thallium influx assay system were assessed to be 8.82 and 0.63, respectively. In a large-scale screening of 98,960 synthetic and natural compounds using the thallium influx assay, 76 compounds displayed consistent KCNQ4 activation, and of these 6 compounds demonstrated EC(50) values of less than 20 μmol/L and 2 demonstrated EC(50) values of less than 1 μmol/L. Taken together, the fluorescence-based thallium flux assay is a highly efficient, automatable, and robust tool to screen potential KCNQ4 openers. This approach may also be expanded to identify and evaluate potential modulators of other potassium channels.
authors.affiliation: The National Center for Drug Screening and The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Qunyi
authors.initials: Q
authors.lastname: Li
authors.forename: Mario
authors.initials: M
authors.lastname: Rottländer
authors.forename: Mingkai
authors.initials: M
authors.lastname: Xu
authors.forename: Claus Tornby
authors.initials: CT
authors.lastname: Christoffersen
authors.forename: Kristen
authors.initials: K
authors.lastname: Frederiksen
authors.forename: Ming-Wei
authors.initials: MW
authors.lastname: Wang
authors.forename: Henrik Sindal
authors.initials: HS
authors.lastname: Jensen
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006801:Humans; D015640:Ion Channel Gating; D051656:KCNQ Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D013793:Thallium / Q000737:chemistry* / Q000378:metabolism
chemical_list: D051656:KCNQ Potassium Channels; C117367:KCNQ4 protein, human; D026902:Potassium Channel Blockers; D013793:Thallium