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.