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

开发单次动作电位发生时死亡的重组细胞系以及作用于离子通道的化合物的新筛选系统

基本信息

批准号：
23659046
负责人：
IMAIZUMI Yuji
金额：
$ 2.41万
依托单位：
Nagoya City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

项目摘要

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

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，通过基因转染突变的人心脏Na^+通道Nav1.5产生了一种新的重组细胞系，其中单动作电位（AP）诱导细胞死亡。 (IFM/Q3)，显示极慢的失活，与野生型内向整流K^+通道、Kir2.1在HEK293细胞中稳定共表达(IFM/Q3+K_<ir>2.1) 在 IFM/Q3+K_<ir>2.1 中，单次电刺激 (ES) 的应用引发了持续超过 30 秒的长 AP，导致细胞死亡超过 70%，而与野生型Nav1.5和K_<ir>2.1共转染的HEK293完全存活。hERG K+通道的额外表达。 IFM/Q3+K_2.1缩短了诱发AP的持续时间，从而显着减少了细胞死亡。用尼非卡兰、E-4031、西沙必利、特非那定和维拉帕米、hERG通道抑制剂处理细胞，恢复了延长的时间。 AP 和剂量依赖性促进 ES 上的细胞死亡。结果表明该细胞系统在 hERG K+ 通道安全测定中具有很高的实用性。为了开发电压门控 Kv1.3 和 Kv1.5 通道阻断剂的筛选系统，共表达 IMF/Q3、K_<ir>2.1 和 Kv1.3 或 Kv1.5 的新细胞系被引入为 IFM/Q3+ K_ <ir>+Kv1.3 和 IFM/Q3+K_<ir>+Kv1.5 分别为 Kv1.3 或 Kv1.5 的共表达。 IFM/Q3+K_<ir> 缩短了诱发的 AP 并防止细胞死亡。在选择性 Kv1.3 阻断剂 margatoxin 存在的情况下，ES 诱导 IFM/Q3+K_<ir>+Kv1.3 中的细胞死亡。但在 IFM/Q3+K_<ir>+Kv1.5 中不存在 4-氨基吡啶（一种非选择性 Kv 通道阻断剂）存在时，ES 应用。在 IFM/Q3+K_<ir>+Kv1.3 中，金合欢素（Kv1.5 阻断剂）和西酞普兰（5-HT 摄取抑制剂）的 IC50 几乎与 IFM 中相同。 /Q3+K_<ir>+Kv1.5 由此发现金合欢素和西酞普兰阻断Kv1.3和Kv1.5。用于 hERG、Kv1.3 或 Kv1.5 通道检测抑制的新细胞系因其简单、准确和高性价比而适合高通量筛选。