Establishment and Characterization of a Stable hERG Cell Line for High-Throughput Drug Cardiac Safety Screening

The hERG potassium channel is critical for cardiac ventricular repolarization and a core target in pre-clinical drug safety screening. A robust, stable cell line with uniform, high hERG expression is essential for high-throughput assessments. In this study, we established a functional stable HEK293T cell line with high hERG expression. The hERG gene was subcloned into Lenti-HA-hERG-P2A-EGFP plasmid, in which GFP serves as a selection marker via a P2A self-cleaving peptide. GFP-positive monoclonal cells were isolated by fluorescence-activated cell sorting (FACS). Confocal imaging confirmed that hERG localized predominantly to the cell membrane, consistent with its physiological role. Manual patch-clamp revealed canonical hERG current properties: a small, stable current during depolarization to 20 mV, followed by a large outward tail current upon repolarization to -40 mV-a hallmark of hERG channel gating. Automated patch-clamp (APC)-based current profiling showed 93.5% of stable hERG cells exhibited peak tail currents > 50 pA (87% > 100 pA, with 49.5% > 400 pA), whereas 100% of blank HEK293T cells showed peak tail currents < 50 pA. Pharmacological validation with E-4031 demonstrated concentration-dependent inhibition of hERG currents, with an IC₅₀ of 29.8 nM, which is consistent with literature-reported values. The stable hERG-expressing HEK293T cell line developed here exhibits consistent hERG expression, canonical channel function, and physiological sensitivity to hERG blockers. When paired with high-throughput APC systems, this cell model provides a robust, standardized platform for pre-clinical drug-induced hERG inhibition evaluation, aiding early detection of long QT syndrome risks and safer drug development.