Dimethyl sulfoxide effects on hERG channels expressed in HEK293 cells.

INTRODUCTION: Dimethyl sulfoxide (DMSO) is widely used as a solvent to facilitate formulation of test substances in cell perfusion solutions. However, DMSO concentration in bath (extracellular) solution is usually limited to 0.1-0.3% to avoid DMSO-induced changes in cell morphology and membrane properties due to elevation of osmolality. The purpose of this study was to examine whether DMSO-induced hyperosmotic effects on hERG expressing cells could be compensated by adding an equivalent amount of DMSO in pipette (intracellular) solution, to investigate DMSO effects on hERG channels, and to determine the impact of DMSO on the potency of hERG channel blockers. METHOD: Whole-cell patch clamp method was used to record hERG currents in HEK293 cells. DMSO at concentrations of 0.1% to 2% was applied to bath and pipette solutions. Various voltage protocols were used to examine DMSO effects on hERG channel properties and to evaluate DMSO impacts on the potency of terfenadine and E-4031. RESULTS: When DMSO was added simultaneously in bath and pipette solutions, normal cell morphology and the proper current recording conditions could be maintained with application of up to 2% DMSO. DMSO slightly shifted the current-voltage relationship, activation curve, and inactivation curve of the hERG channel to more positive voltages. DMSO had little effect on the concentration-response relationship of hERG channel blockers we assessed. The IC50 for terfenadine and E-4031 were not significantly changed in the presence of 0.3, 0.5, 1 and 2% DMSO. DISCUSSION: Our results demonstrate that changes in cell morphology induced by extracellular DMSO can be prevented by application of DMSO in pipette solution. By utilizing this approach, we successfully performed hERG current recordings using bath solution containing up to 2% DMSO. DMSO-induced shifts of the voltage-dependence of hERG channel gating had little impact on the potency of hERG channel blockers.

Estimation of potency of HERG channel blockers: impact of voltage protocol and temperature.

INTRODUCTION: The HERG channel is widely used for the assessment of proarrhythmic risk for new drugs. HERG channel blockers obstruct channel functions through various mechanisms, which usually show time dependence, voltage dependence, and state dependence. The voltage protocol and temperature may affect the estimation of drug potency, but limited information is available in this regard. The purpose of this study was to evaluate the influence of voltage protocol and temperature on predicting the potency of HERG channel blockers, and to determine electrophysiological approaches for new drugs screening studies. METHOD: Whole-cell patch-clamp electrophysiology was carried out by utilizing different voltage step protocols to examine the potency of compounds known to preferentially block the channel in the closed (ketoconazole and BeKm-1), open, and/or inactivated states (E-4031, astemizole, and terfenadine) in HEK293 cells transfected with HERG cDNA at room temperature and near-physiological temperature. RESULTS: Drug potency determined using different voltage protocols varied dependent on the mechanisms of drug actions. For most compounds, the IC(50) values obtained with a long pulse step protocol at room temperature were close to those determined with the voltage protocols designed to disclose their intrinsic potency. Relative to room temperature, the potency of E-4031, terfenadine, and ketoconazole was not changed at approximately 35 degrees C, but potency of astemizole was reduced. DISCUSSION: The long pulse step protocol with room temperature can be selected for HERG channel safety screening studies. Alternative voltage protocols or temperatures should be considered if HERG study results are not consistent with other cardiac safety assessments.