| Assay Method Information | |
| | Human ERG (hERG) assay |
| Description: | The whole-cell patch-clamp technique was used to investigate the effects of the test items on hERG (human-ether-a-go-go related gene) potassium channels in stably transfected CHO cells near physiological temperature (36±1° C.). The effects of compounds on hERG K+-current parameters were evaluated at 4 concentrations (0.3-3-30-300 μM) in at least 3 CHO cells stably expressing the hERG channel. For electrophysiological measurements cells were seeded onto 35 mm sterile culture dishes containing 2 ml culture medium without Hygromycin B. Cells were cultivated at a density that enabled single cells (without visible connections to neighbouring cells) to be measured. Cells were incubated at 37° C. in a humidified atmosphere with 5% CO2 (rel. humidity about 95%). The cells were continuously maintained in and passaged in sterile culture flasks containing nutrient mixture F-12 (DMEM/F-12 with L-Glutamine) supplemented with 10% foetal bovine serum and 10% penicillin/streptomycin solution. Every day at least three cells were treated with a selective IKr blocker (E-4031, reference substance) to assure accuracy of the method. The 35 mm culture dishes upon which cells were seeded at a density allowing single cells to be recorded were placed on the dish holder of the microscope and continuously perfused (at approximately 1 ml/min) with the bath solution (sodium chloride 150 mM, potassium chloride 4 mM, calcium chloride 1.2 mM, magnesium chloride 1 mM, HEPES 10 mM, pH (NaOH) 7.4) at near physiological temperature (36±1° C.). After formation of a Gigaohm seal between the patch electrodes and individual hERG stably transfected CHO cells (pipette resistance range: 2.0 MΩ-7.0 Mω; seal resistance range: >1 Gω) the cell membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-cell patch-configuration). In case the quality of the seal was poor, the process of seal formation was repeated with a different cell and a new pipette. As soon as a stable seal was established, hERG outward tail currents were measured upon depolarization of the cell membrane to −40 mV for 50 ms followed by 500 ms at +20 mV (activation of channels) from a holding potential of −80 mV and upon subsequent repolarization to −40 mV for 500 ms. This voltage protocol was run at least 10 times at intervals of 10 s. If current density was judged to be too low for measurement, another cell was recorded. Once control recordings have been accomplished, cells were continuously perfused with a bath solution containing the test items. During wash-in of the test item the voltage protocol indicated above was run continuously again at 10 s intervals until the steady-state level of block was reached. The four test item concentrations of the compound were applied sequentially to 3 cells in a cumulative manner. As hERG tail currents were inhibited by the test item, the concentration-response curve was generated and ICso value calculated. Based on the IC50 value the IC20 was estimated. |
| Affinity data for this assay | |
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