The experimental protocol was approved as “a non-animal study” in advance by the Ethics Review Committee for Animal Experimentation of Oita University School of Medicine.
2.1. Cell Preparation
The human embryonic kidney 293 (HEK293) cells stably expressing the hERG channel (hERG-HEK293) are generous gifts from Professor Imaizumi, Nagoya City University, Japan. hERG-HEK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA) with 15% fetal bovine serum (FBS, Gibco) at 37°C and exposed to an atmosphere of 5% CO2. The culture medium was also supplemented with 400 µg·mL-1 gentamycin (G418, Calbiochem, USA). hERG-HEK cells were treated with acute- (5 min) and long-term (24 h ) perfusion of gemcitabine for detection of the drug actions on IhERG and the hERG channel gating properties.
2.2. Electrophysiological recording
We used a whole-cell patch clamp technique for the measurement of IhERG according to the previous studies [15-17]. Briefly, IhERG was recorded by whole-cell patch clamp using an EPC-9 amplifier controlled by Pulse ver.8 software (HEKA Eletronik, Lambrecht, Germany). Patch pipettes were pulled from 75-mm plain capillary tubes (Drummond Scientific Co., Broomall, PA, USA) by Model P-97 (Sutter Instrument Co., Novato, CA, USA), and were heat-polished subsequently to achieve the pipette resistance at 2–4 MΩ when filled with the pipette solution shown below. Series resistance was compensated by at least 80% and was continually monitored throughout the experiment. All the current measurements were done at room temperature (20–23°C). For the current recording, the chamber was filled with bath solution contained (mM) NaCl 135, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, glucose 10 (pH 7.4 by NaOH). The patch electrodes were filled with pipette solution consists of (mM) KCl 135, MgCl2 1, EGTA 5 and HEPES 10 (pH 7.2 by KOH). For analysis of the conductance-voltage relationship or IhERG.tail-V relationship, tail current of IhERG was evoked through a 4-s voltage step to -40 mV, following a 4-s depolarization step with a 10 mV stepwise increase from -80 to 60 mV, which was initiated after a holding potential of -80 mV (Figure 1). The peak of the outward tail current of IhERG were plotted against the voltage of test pulses, and the data were fitted to the Boltzmann function, as: f(V) = 1/(1 + e[(V1/2−V)/k]) + C, where the V1/2 value is the membrane potential when the relationship reaches half level, and the k value is the slope factor. C is a constant component. For analysis of the activation gating properties, we used an envelope of tails procedure, which takes advantage of the fact that although at potentials positive to approximately +30 mV a small and brief transient component of inactivation overlaps with activation , recovery from inactivation is rapid  and deactivation is relatively slow . The activating kinetics were evaluated by plotting the plotted the activating inward tail current envelope as a function of test pulse duration by use of a single exponential equation fits, which provided the time constant (τact) for activation at each individual potential, based on the simple Hodgkin and Huxley model  (Scheme I). Namely, hERG-HEK cells were evoked through a depolarization step, from -10 mV to +30 mV, for an incremental duration for 20ms-4,000 ms followed by a holding potential of -100 mV. At each depolarizing potential, τact was then plotted against the depolarizing voltage, yielding τact-voltage relationship (Figure 3). For analysis of the inactivation gating properties, the inactivating kinetics were evaluated by the time constant of the tail current; hERG-HEK cells were evoked through a 2.5-s depolarizing steps (test potentials) from -60 mV to +60 mV to inactivate, following a 5-ms repolarization at -120 mV from the holding potential of +60 mV (Scheme I). At each test potential, onset of each inactivation current or the decay of tail current during each voltage step was fitted by use of a single exponential curve, which provided the time constant for inactivation (τinact) at the individual potentials, then τinact was plotted against the voltage, yielding τinact-voltage relationship (Figure 4). For analysis of the deactivation gating properties, the deactivating kinetics were evaluated by fitting the deactivating inward tail current at each repolarizing voltage step (-180 mV to -110 mV) from the holding potential of +60 mV to a single exponential curve (Scheme I), providing the time constant for deactivation (τdeact) at the individual potentials, then τdeact was plotted against the voltage, yielding τdeact-voltage relationship (Figure 5). For analysis of the reactivation gating properties namely recovery from the inactivation gating properties, the reactivating kinetics were evaluated by the amplitude of peak tail current corresponding to the hyperpolarizing pulse from -200mV to +60 mV for 5 ms to allow inactivation to recovery to a steady-state (Scheme I), followed by a return step to +60mV (Figure 6).
The peak of the tail currents plotted as a function of the preceding test pulse potentials were fitted with a Boltzmann function, namely the steady-state inactivation curve, was assessed as the reactivation kinetics. At more negative voltages from -170 mV to -200 mV, a proportion of the hERG channels also deactivate during the 5 ms test potential, leading the observed decrease in the peak IhERG at negative potentials. Therefore, before fitting of the Boltzmann equation, this deactivation was corrected based on the pulse protocol in Figure 5. For analysis of the concentration dependency of gemcitabine on IhERG, we used six concentrations of gemcitabine (0.01, 0.05, 0.1, 1, 5, and 50 µM) to construct the half-maximal inhibitory concentration (IC50) curve, which was sigmoidal in shape plotted against the log [gemcitabine] fitted with the Hill equation.