| Assay Method Information | |
| | Experimental Evaluation of the In Vitro Inhibitory Effect (IC50) of hERG |
| Description: | Stabilized cells were dropped onto circular slides and placed in a culture dish with a cell density below 50% and incubated overnight. Cells for experiments are transferred to a bath of approximately 1 ml embedded in an inverted microscope stage and perfused with extracellular fluid at a rate of 2.7 ml/min. The experiment can be started after 5 minutes of stabilization. Membrane currents were recorded using a HEKA EPC-10 membrane clamp amplifier and a PATCHMASTER acquisition system (HEKA Instruments Inc., D 67466 Lambrecht, Pfalz, Germany). All experiments were performed at room temperature (22 24 C.). A P-97 microelectrode puller (Sutter Instrument Company, One Digital Drive, Novato, Calif. 94949) was used to straighten the electrodes (BF150-110-10) in the experiments. The electrode had an inner diameter of 1-1.5 mm and an inlet resistance of 2-4 MΩ when filled with internal fluid. hERG potassium channels were electrophysiologically stimulated by first clamping the membrane voltage at −80 mV, giving the cells a continuous 2 s, +20 mV voltage stimulation to activate the hERG potassium channels, and then repolarizing to −50 mV for 5 s to generate an outward tail current with a stimulation frequency of every 15 s. Current values are the peak tail currents.Channel currents were recorded in whole-cell recording mode in the experiments. First, the extracellular fluid (approximately 2 mL per minute) was perfused and recorded continuously, and the current was stabilized (Run-Down less than 5% for 5 minutes), at which point the peak tail current was the control current value. Next, the extracellular fluid containing the drug to be tested was instilled and recorded continuously until the inhibitory effect of the drug on the hERG current reached a steady state, at which point the peak tail current was the post-drug current value. The criterion for steady state was determined by the coincidence of the three most recent consecutive current recording lines. After reaching steady state, if the hERG current returned to or approached the size before the drug was added after washout with extracellular fluid, the test could be continued with other concentrations or drugs. 30 μM Quinidine (Quinidine) was used as a positive control in the experiment to ensure that the cells used responded normally. |
| Affinity data for this assay | |
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