Automated patch-clamp assay

hERG activity was examined in the population-patch-clamp mode (PPC) of IonWorks Quattro™ (Molecular Devices, LLC, Sunnyvale, CA, USA), an automated patch clamp instrument. Compound effects were tested using dual compound additions at 1 μmol/L and 10 μmol/L. The CHO-K1 cells stably expressing hERG channels were freshly dislodged from flasks with Trypsin-EDTA (0.05%) for 4 min, spin down twice at 700×g, and suspended in the extracellular solution (in mmol/L) with 1.8×10⁶ per mL: 137 NaCl, 4 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 10 Glucose, pH 7.4 adjusted with NaOH and dispensed into a 384-well population patch clamp (PPC) plate. The cell plating density was 6300 cells/well.

After dispensing, seal resistance of cells was measured for each well and cells were perforated by incubation with 50 μg/mL amphotericin B (Sigma, St Louis, MO, USA) in the internal solution composed of (in mmol/L): 40 KCl, 100 K-Gluconate, 1 MgCl₂, 5 HEPES, 2 CaCl₂, pH 7.2 adjusted with KOH. Activity of hERG was then measured with the recording protocol as shown in Figure 1A. hERG currents were evoked by two voltage pulses with a 3 s interval. The voltage pulse consisted of a 100 ms step to −30 mV, a conditioning pre-pulse (2 s duration, +25 mV or +45 mV) followed by a test pulse (2 s duration, −30 mV) from a holding potential at −70 mV. After a 3 s interval at −70 mV, a second pulse protocol was applied consisting of a 100 ms step to −30 mV, a pre-pulse (2 s duration, +45 mV) followed by a test pulse (2 s duration, −30 mV). Leak currents were linearly subtracted extrapolating the current elicited by a 100 ms step to −80 mV from a holding potential of −70 mV. Positive controls (1 μmol/L dofetilide) and negative controls [external buffer with 0.02% (first addition) and 0.2% (second addition) (v/v) DMSO] values were measured in each plate. Compound incubation duration was 3 min for both the first addition (1 μmol/L) and second addition (10 μmol/L). Z′ factor was calculated to evaluate data quality. Wells with a peak tail current amplitude bigger than 0.2 nA, a seal resistance more than 30 Mohms, and reduction of seal resistance less than 25% were included for data analysis.

Seal resistance and current expression of hERG-CHO stable cell line measured using the IonWorks Quattro^™ system. (A) Recording protocols and the typical hERG current traces in the absence and presence of 1 μmol/L Dofetilide. In the displayed protocol, the solid line represented the protocol used for majority of compounds while the dashed line presented a voltage pulse used in a small fraction of compounds of the screening. Dose-response curve for Dofetilide is displayed in the right panel. (B) Histograms of seal resistance recorded across 384 wells of single-hole (HT) (black color filled bars) and population patch clamp (PPC) (grey color filled bars) using hERG-CHO cells at a density 1.8×10⁶ cells/mL. The bootstrap analysis was applied to the data from single-hole mode to simulate the distribution of seal resistance in the PPC mode (as shown in the unfilled bars). (C) The distribution of the seal resistance (PPC mode) for the screened library with the mean±SD values at 89.66±36.26 Mohms. (D) Histograms of peak tail current amplitude obtained from single-hole (black color filled bars) and PPC modes (grey color filled bars). Peak currents at the first test pulse were measured in each well. Similar to the seal resistance, the bootstrap analysis was performed to estimate the distribution of hERG tail current on PPC mode using experimental results from the single-hole mode (as shown in unfilled bars). (E) The distribution of the peak currents (PPC mode) for the screened library with the mean±SD values at 0.62±0.17 nA.

Peak amplitudes of tail currents and steady-state currents from the second pulse were measured before and after compound treatment. Compound effects were assessed by percentage changes of hERG peak tail currents and steady-state currents, which were calculated by dividing the difference between pre- and post-compound hERG currents by the respective pre-compound currents in the same well. No corrections for liquid junction potentials (estimated as −20 mV by comparing the hERG tail current reversal potential with the calculated Nernst potential for potassium ion) were applied. The current signal was sampled at 0.625 kHz.