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

Two-pore domain K (K) channels are thought to be druggable targets. However, only a few agents specific for K 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 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-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 channel blockers enhanced cell death. IC 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 channels can be accurately assessed with high efficiency and throughput by this novel HTS system.