Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background Two-Pore Domain K+ Channels. (July 2019)

• Record Type: Journal Article
• Authors: Kawasaki, Keisuke; Suzuki, Yoshiaki; Yamamura, Hisao; Imaizumi, Yuji
• Is Part Of: SLAS discovery. Volume 24:Number 6(2019)
• Page Start: 641
• Page End: 652
• Publication Date: 2019-07
• Subjects: ion channel -- cell-based assays -- high-throughput screening -- membrane potential -- two-pore domain K+ channel; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; Biomolecules -- Analysis; Drugs -- Analysis; Drugs -- Testing; Drug Evaluation, Preclinical; Molecular Biology -- methods; Periodicals; Periodicals; 615.1
• DOI: 10.1177/2472555219829745
• Languages: English
• ISSNs: 2472-5552

Abstract

Two-pore domain K + (K2P ) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based "test cell" expressing a mutated Na + channel (Nav1.5) with markedly slowed inactivation, as well as a K + channel (Kir2.1) that sets the membrane potential quite negative, close to K + equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba 2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na + influx. When either the TWIK-related acid-sensitive K + (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba 2+ -induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.