Effects Of Fluconazole On Electrophysiological Functions Of HERG Channel

ObjectiveTo investigate the effects of the fluconazole on wild type-hERG (WT-hERG), Y652A-hERG, and F656C-hERG currents expressed in human embryonic kidney (HEK293) cells, and mechanism of block of the hERG K+ Channel by fluconazole.Methods1 WT-hERG were transfected into HEK293 cells by calcium phosphate transfection method. hERG currents were recorded by a whole-cell patch-clamp technique.2 Y652A-hERG and F656C-hERG were transfected into HEK293 cells, to record the effects of fluconazole on the hERG potassium currents.Results1 Fluconazole inhibited the amplitudes of the currents at the end of voltage steps, and the tail currents of WT-hERG in concentration-dependent manner. Half-maximum block concentrations (IC50) were 48.2±9.4μM.2 Fluconazole did not change other channel kinetics (activation and steady-state inactivation) of WT-hERG channel. In the absence of fluconazole and in the presence of 10μM fluconazole:The derived mean V1/2 and k values were:control V1/2=-3.9±0.3 mV; fluconazoleV1/2=-6.3±0.5 mV(P>0.05, n=9), control k=10.4±0.3 mV; fluconazole k=10.3±0.4 mV(P>0.05, n=9), with the IhERG activation relations in control and fluconazole closely overlying one another, In control and fluconazole the inactivation V1/2 values were respectively,-43.3±0.8 mV and -43.1±0.9 mV (P>0.05, n=9), with corresponding k values of 22.9±0.6 mV and 23.6±0.8 mV (P>0.05, n=9), fluconazole did not alter the voltage-dependence of IhERG inactivation.3 Y652A and F656C mutations largely inhibited a blockade of hERG currents by compared with WT-hERG in the presence of 300μM fluconazole. The blockades of WT-hERG and Y652A-hERG currents were 61.1±2.7% and 22.8±5.6%, respectively. The blockades of WT-hERG and F656C-hERG were 41.7±3.7% and 29.8±6.3%, respectively. Thus, fluconazole had significant effect on either Y652A-hERG or F656C-hERG currents by compared with WT-hERG(P<0.01, ANOVA).Conclusions1 Fluconazole produced concentration dependent (not frequency dependent) inhibition effect on WT-hERG channel currents to HEK293 cells.2 The S6 mutants Y652A and F656C significantly attenuate WT-hERG channel blockade by fluconazole, these results indicate that both Y652 and F656 are critical sites for the interaction between fluconazole and the hERG channels.