Development of a Novel Cell-Based Assay System for High-Throughput Screening of Compounds Acting on Background...

Two-pore domain K⁺ (K_2P) channels are thought to be druggable targets. However, only a few agents specific for K_2P 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 Ba²⁺ 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, Ba²⁺-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K_2P 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 K_2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.