钾通道调节剂的高通量筛选模型  

High throughput screening method of potassium channel regulators

作　　者：潘雅萍[1] 徐向华[1] 王晓良[1] 

机构地区：[1]中国医学科学院,中国协和医科大学药物研究所,北京100050

出　　处：《药学学报》2004年第2期85-88,共4页Acta Pharmaceutica Sinica

基　　金：国家"973计划"项目资助 (G1 9980 51 1 0 6 );"86 3计划"项目资助 ( 2 0 0 1AA2 34 0 2 1 )

摘　　要：目的　建立钾通道调节剂高通量筛选的细胞模型。方法　 96孔板上细胞负载荧光染料DiBAC4(3 ) ,测定不同化合物对荧光强度的影响 ,反映细胞膜电位的变化 ,间接反映化合物对钾通道的作用。结果　高钾去极化和钾通道阻断剂 4 AP ,TEA ,E 40 3 1,glibenclamide ,quinidine和nifedipine均能增强细胞的荧光强度 ,钾通道开放剂cromakalim能减弱细胞的荧光强度 ,上述化合物在一定剂量范围内均有剂量效应关系。利用该模型筛选 76个化合物 ,发现 9个化合物的荧光强度变化值有较好的剂量效应关系 ,有待膜片钳技术的进一步验证和筛选。结论　此方法简单 ,易于操作 ,重现性好 。Aim To discover new regulators of potassium channel, an in vitro assay based on DiBAC 4(3) to determine the fluorescence was established for high throughput sc reening. Methods A cell-based 96-well format fluorescence assay using DiBAC 4(3) in cultured P C12 cells was described. Cells were loaded with 5 μmol·L -1 DiBAC 4( 3) and incubated at 37 ℃ for 30 min before adding KCl or several known pota ssium channel regulators. The cellular DiBAC 4(3) fluorescence responce was the n detected. The fluorescence changes can be used to evaluate membrane potential changes, which are determined mainly by potassium channels. Results Extracellular high K +-induced depolarization and several potassium channel bl ockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine al l evoked increases in DiBAC 4(3) fluorescence responce. The potassium channel o pener, cromakalim, evoked decrease in DiBAC 4(3) fluorescence responce. The flu orescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were i n a concentration-dependent manner. In 76 compounds screened by using the estab lished DiBAC 4(3)-based assay, 9 compounds were found to change the fluorescen ce dose-dependently. Patch clamp technique is needed to further testify and scr een their actions on potassium currents. Conclusion The DiBAC 4(3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulator s.

关 键 词：钾通道调节剂 高通量筛选 细胞模型 细胞膜电位 荧光染料 

分 类 号：R972[医药卫生—药品] R965.1[医药卫生—药学]