Hexachlorophene Is a Potent KCNQ1/KCNE1 Potassium Channel Activator Which Rescues LQTs Mutants

Zheng, Yueming; Zhu, Xuejing; Zhou, Pingzheng; Lan, Xi; Xu, Haiyan; Li, Min; Gao, Zhaobing

Cell culture and transfection CHO cells were grown in 50/50 DMEM/F-12 (Gibco) with 10% fetal bovine serum (FBS), and 2 mM L-glutamine (Invitrogen). To express the channels and mutants, cells were split at 24 h before transfection, plated in 60-mm dishes, and transfected with Lipofectamine 2000 TM reagent (Invitrogen), according to the manufacturer's instructions. A GFP cDNA (Amaxa, Gaithersburg, MD) was cotransfected to identify the transfected cells by fluorescence microscopy. FluxOR thallium assay CHO cells stably expressing the rat KCNQ2 were routinely cultured in DMEM/F12 medium, supplemented with 10% FBS and 500 mg/mL G418. The FluxOR thallium assay protocol was the manufacturer's protocol. CHO-KCNQ2 cells were seeded in wells of 96-well plates at ,10,000 cells/well and grown until 80-90% confluence at 37uC in a 5% CO 2 incubator. The medium was removed the following day and 80 mL of FluxOR loading buffer was added to each well for 90 min at room temperature (RT) in darkness. After removing the loading buffer, 100 mL/well of assay buffer and 20 mL/well of 7X control/test compound were added to cells at RT in darkness. Compounds to be tested were prepared using assay buffer; controls were assay buffer (EC 0 ), EC 50 of Retigabine and EC 100 of ztz240. After 30 min, cell plates were loaded on FDSS. After 10 seconds of recording, 20 mL/well of stimulus buffer was added. The plates were read every second for 110 s. The stimulus buffer contained 1.30 mM K2SO4 and 9.80 mM Tl 2 SO 4 . The gpotentiation% ((R test -R control )/(R control -R buffer )*100%) was calculated for each well using the 35 second fluorescence ratio. To identify compounds with potentiation activity on KCNQ channels, a thallium flux assay was developed and used to screen a Microsource TM library of 1,280 compounds at 10 mM final concentration. In the pilot screening, HCP exhibited strong potentiation on the fluorescence signal of KCNQ2.