title: 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.
authors.affiliation: Nagoya City University, Nagoya, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Fujii
authors.forename: Susumu
authors.initials: S
authors.lastname: Ohya
authors.forename: Hisao
authors.initials: H
authors.lastname: Yamamura
authors.forename: Yuji
authors.initials: Y
authors.lastname: Imaizumi
mesh_terms: D000200:Action Potentials / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D020117:Cisapride / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D051638:Ether-A-Go-Go Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000009:adverse effects*; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D015222:Sodium Channels / Q000235:genetics; D016593:Terfenadine / Q000494:pharmacology; D014162:Transfection; D014700:Verapamil / Q000378:metabolism
chemical_list: D051638:Ether-A-Go-Go Potassium Channels; C489457:KCNJ2 protein, human; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010880:Piperidines; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying; D011725:Pyridines; D011744:Pyrimidinones; C568320:SCN5A protein, human; D015222:Sodium Channels; C063968:E 4031; C076259:nifekalant; D016593:Terfenadine; D014700:Verapamil; D020117:Cisapride