[High throughput screening method of potassium channel regulators].
OBJECTIVE
To discover new regulators of potassium channel, an in vitro assay based on DiBAC4 (3) to determine the fluorescence was established for high throughput screening.


METHODS
A cell-based 96-well format fluorescence assay using DiBAC4 (3) in cultured PC12 cells was described. Cells were loaded with 5 mumol.L-1 DiBAC4 (3) and incubated at 37 degrees C for 30 min before adding KCl or several known potassium channel regulators. The cellular DiBAC4 (3) fluorescence responce was then 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 blockers including 4-AP, TEA, E-4031, glibenclamide, quinidine and nifedipine all evoked increases in DiBAC4 (3) fluorescence response. The potassium channel opener, cromakalim, evoked decrease in DiBAC4 (3) fluorescence response. The fluorescence changes of 4-AP, TEA, glibenclamide, nifedipine and cromakalim were in a concentration-dependent manner. In 76 compounds screened by using the established DiBAC4 (3)-based assay, 9 compounds were found to change the fluorescence dose-dependently. Patch clamp technique is needed to further testify and screen their actions on potassium currents.


CONCLUSIONS
The DiBAC4 (3)-based assay is easily operated, economical and repeatable. So, it can be performed by high throughput screening for potassium channel regulators.
Pan
Ya-ping
YP
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Xu
Xiang-hua
XH
Wang
Xiao-liang
XL
D015761:4-Aminopyridine / Q000494:pharmacology; D000818:Animals; D001463:Barbiturates / Q000737:chemistry*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D019806:Cromakalim / Q000494:pharmacology; D007555:Isoxazoles / Q000737:chemistry*; D008564:Membrane Potentials / Q000187:drug effects; D009543:Nifedipine / Q000494:pharmacology; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects*; D011725:Pyridines / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D051381:Rats
D001463:Barbiturates; D002121:Calcium Channel Blockers; D007555:Isoxazoles; D010880:Piperidines; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D011725:Pyridines; D019806:Cromakalim; C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; C063968:E 4031; D015761:4-Aminopyridine; D009543:Nifedipine; D011802:Quinidine