Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay

• Published in: Analytical biochemistry 2011-11, Vol.418 (1), p.66-72
• Main Authors: Li, Qunyi, Rottländer, Mario, Xu, Mingkai, Christoffersen, Claus Tornby, Frederiksen, Kristen, Wang, Ming-Wei, Jensen, Henrik Sindal
• Format: Article
• Language: English
• Subjects: 86Rubidium flux; Animals; CHO Cells; Cricetinae; Drug Evaluation, Preclinical - methods; electrophysiology; Fluorescence; High-throughput screening; High-Throughput Screening Assays; Humans; Ion Channel Gating; KCNQ; KCNQ Potassium Channels - chemistry; KCNQ Potassium Channels - metabolism; Kv7; Patch clamp; Patch-Clamp Techniques; permeability; Potassium Channel Blockers - pharmacology; Potassium channel opener; potassium channels; screening; Stable cell line; thallium; Thallium - chemistry; Thallium - metabolism; Thallium flux
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2011.06.040

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 EC50 values of the positive control compound (retigabine) determined by the thallium and 86rubidium 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 EC50 values of less than 20μmol/L and 2 demonstrated EC50 values of less than 1μmol/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.