Patch-clamp electrophysiology
Automated patch-clamp recordings were performed using a SyncroPatch 384PE (Nanion Technologies, Munich, Germany), with NPC-384 single hole medium resistance (4-5 MΩ) chips as described previously (16). Prior to experiments, stably expressing hERG-CHO cells were dissociated with Accumax (Sigma-Aldrich, Sydney, Australia) and resuspended to a density of ~180 - 440K cells/mL in chip fill solution, containing (in mM): 140 NaCl, 5 KCl, 10 HEPES, 5 Glucose, pH 7.4 with NaOH (~298 mOsm). Cells were incubated at 4oC for at least 1 hour prior to experiments. For whole cell recordings, the intracellular solution contained (in mM): 110 KF, 10 KCl, 10 NaCl, 10 HEPES, 10 EGTA, pH7.2 (~285mOsm) and the standard extracellular solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 5 Glucose, pH 7.4 with NaOH (~298 mOsm). To assess the impact of electrolyte changes on drug block, extracellular KCl or MgCl2 concentrations were varied from 3 to 7 mM and 0.5 to 3 mM, respectively. For pH experiments, the pH was adjusted to pH 7.0 or 6.5. Cells were voltage clamped at a holding potential of -80 mV then depolarized to 40 mV for 4 s followed by a 1 s test pulse to -40 mV to elicit hERG tail currents, with 15 s start-to-start interval. The degree of drug block of hERG current was assessed as the change in peak amplitude of the tail current after 12 minutes of drug application.