Development of Recombinant Cell Line Co-expressing Mutated Nav1.5, Kir2.1, and hERG for the Safety Assay of Drug Candidates

Abstract

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K + channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na + channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K + channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K + channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC 50 values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K + channel safety assay.

New Screening System for Selective Blockers of Voltage-Gated K+ Channels Using Recombinant Cell Lines Dying Upon Single Action Potential

“…In contrast, the end current was markedly inward in the presence of 300 mM 4-AP, a non-selective Kv-channel blocker. This finding indicates that the Na + current through IFM/Q3 mutated Na + channels had a substantially sustained component, as has been reported (14) (Fig. 1Aa).…”

“…However, the addition of hERG-blocking compounds, including nifekalant, E-4031, cisapride, terfenadine, and verapamil, markedly prolongs action potentials upon ES and results in cell death. The quantitative analyses of hERG-blocking effects of test compounds can be performed in this simple assay system (14). The technology developed in the previous study has been applied to patent filings (PCT/ JP2011/064967), and the laid-open disclosure number is WO2012/002460.…”

“…Cell viability was monitored by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay as described previously (14). MTT was dissolved in phosphate-buffered saline at 5 mg/ml and filtered to sterilize it and remove a small amount of insoluble residue present in some batches of MTT.…”

“…The cell death was measured with a simple and popular assay, such as the MTT method (14). For the practical use of this system for the hERG safety assay, hERG channels have been additionally co-expressed (IFM/Q3+Kir+hERG).…”

“…NM_198056.2) as described previously (14). Mutated Nav1.5 (IFM/Q3) was ligated into a mammalian expression vector, pcDNA3.1/Neo(+) (Invitrogen, Carlsbad, CA, USA), using the TaKaRa ligation kit version 1 (TaKaRa, Osaka).…”