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

为了提供一种简单但高通量的筛选作用于离子通道的化合物的方法，通过基因转染法获得了一种新的重组细胞系，在该细胞系中，单动作电位(AP)诱导了细胞死亡。突变的人心肌钠离子通道NaV1.5(IFM/Q3)和野生型内向整流钾通道Kir2.1在HEK293细胞(IFM/Q3+K_&lt；IR&gt；2.1)中稳定共表达。在IFM/Q3+K&lt；IR&gt；2.1中，单次电刺激(ES)可诱导30个以上的长AP，并导致细胞死亡70%以上，而野生型NaV1.5与K&lt；IR&gt；2.1共转染的HEK293细胞完全存活。在IFM/Q3+K&lt；IR&Gt；2.1中额外表达Herg K+通道可缩短诱发AP的持续时间，从而显著减少细胞死亡。HERG通道阻断剂硝苯卡兰、E-4031、西沙必利、特非那定和维拉帕米可使延长的AP恢复，并呈剂量依赖性地促进ES细胞死亡。结果Indica…进一步证明了该细胞系统在HERG K+通道安全检测中的高实用性。此外，为了建立电压门控Kv1.3和Kv1.5通道阻滞剂的筛选系统，引入了共表达IMF/Q3、K_&lt；IR&gt；2.1和Kv1.3或Kv1.5的新细胞系IFM/Q3+K_&lt；IR&gt；+Kv1.3和IFM/Q3+K_&lt；IR&gt；+Kv1.5。Kv1.3或Kv1.5共表达于IFM/Q3+K&gt；IR和Gt；可缩短诱发的AP，防止细胞死亡。ES可诱导IFM/Q3+K+Kv1.3细胞死亡，但不能诱导IFM/Q3+K&gt；IR+Kv1.5细胞死亡。在非选择性Kv通道阻滞剂4-氨基吡啶存在的情况下，ES诱导两种细胞系的细胞死亡。在IFM/Q3+K+Kv1.3和IFM/Q3+K_&lt；IR&gt；+Kv1.5中，Kv1.5阻滞剂Aacacetin和5-羟色胺摄取抑制剂西酞普兰的IC50值与IFM/Q3+K_&gt；IR&gt；+Kv1.5几乎相同。结果发现，金合欢素和西酞普兰对Kv1.3和Kv1.5均有阻断作用，且无明显选择性。用于HERG、Kv1.3或Kv1.5通道抑制实验的新细胞系，由于其简单、准确和高性价比，适合于高通量筛选。较少

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