Development of recombinant cell lines dying upon single action potentialoccurrence and the new screening system for compounds acting on ion channels

点击翻译按钮获取中文摘要

To provide a simple but high throughput screening method for compounds acting on ion channels, 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+K_<ir>2.1). In IFM/Q3+K_<ir>2.1, application of single electrical stimulation (ES) elicited a long AP lasting over 30 s and led cells to die by over 70 %, while HEK293 co-transfected with wild type Nav1.5 and K_<ir>2.1 fully survived. The additional expression of hERG K+channels in IFM/Q3+K_<ir>2.1 shortened the duration of evoked AP and, thereby, markedly reduced the cell death. The treatment of the cells with nifekalant, E-4031, cisapride, terfenadine and verapamil, hERG channel inhibitors, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. Results indica … More te the high utility of this cell system for hERG K+channel safety assay. Moreover, to develop a screening system for blockers of voltage-gated Kv1.3 and Kv1.5 channels, new cell lines co-expressing IMF/Q3, K_<ir>2.1 and Kv1.3 or Kv1.5 were introduced as IFM/Q3+K_<ir>+Kv1.3 and IFM/Q3+K_<ir>+Kv1.5, respectively. Co-expression of Kv1.3 or Kv1.5 to IFM/Q3+K_<ir> shortened the evoked APs and prevented the cell death. In the presence of margatoxin, a selective Kv1.3 blocker, ES induced the cell death in IFM/Q3+K_<ir>+Kv1.3, but not in IFM/Q3+K_<ir>+Kv1.5. In the presence of 4-aminopyridine, a non-selective Kv channel blocker, ES application elicited cell death in both cell lines. The IC50s of acacetin, a Kv1.5 blocker, and citalopram, a 5-HT uptake-inhibitor, in IFM/Q3+K_<ir>+Kv1.3 were almost identical to those in IFM/Q3+K_<ir>+Kv1.5. It was, thereby, found that acacetin and citalopram block both Kv1.3 and Kv1.5 without significant selectivity. The new cell lines for hERG, Kv1.3 or Kv1.5 channel inhibition assay fit to the high-throughput screening because of its simplicity, accuracy and high cost-performance. Less