Representative current recordings in control and 3 min after application of β-blockers in the same cell are shown in Figure 2. HERG current was elicited from a holding potential of −80 mV by 4-s long depolarising steps to between −70 and 60 mV applied in 10 mV increments every 15 s. Tail current was recorded with a step pulse to −50 mV for 6 s. Carvedilol, propranolol, and ICI 118551 reduced HERG current amplitude during the depolarising step as well as tail current at relatively low concentrations. High concentrations of metoprolol were required to reduce HERG current. However, atenolol did not produce noticeable inhibition of HERG current when used at concentrations up to 1 mM.
The effects of metoprolol, carvedilol, propranolol and ICI 118551 on the HERG current–voltage (I–V) relationship are shown in Figure 3. The averaged normalised I–V relationships for HERG current measured at the end of the depolarising steps and for tail current are shown. The control HERG current during the depolarising steps was maximal at about 0–10 mV, with tail current fully activated following steps to 10–20 mV. Metoprolol (100 μM), carvedilol (1 μM), propranolol (10 μM) and ICI 118551 (10 μM) reduced the steady-state current amplitude to 48.9±10% (n=5), 34.6±8.6% (n=4), 32.1±6.4% (n=5) and 55.0±3.5% (n=7) of the control at 0 mV, respectively, and the peak tail current amplitude to 54.3±6.1% (n=5), 41.1±8.3% (n=4), 44.3±2.1% (n=5) and 61.6±3.8% (n=7) of the control at 20 mV, respectively. Voltage-dependence of activation (Figure 3) was also evaluated by plotting normalised tail current as a function of voltage. Normalised data were fitted with a Boltzmann function:
where Imax is maximum amplitude, V1/2 and κ are half-activation voltage and the slope factor, respectively. After application of the test drugs, the half-maximal activation voltages significantly shifted in a negative direction compared with the control (metoprolol −13.2±1.5 to −17.0±3.8 mV, P<0.05, n=5; carvedilol −11.0±1.3 to −21.3±1.8 mV, P<0.05, n=4; propranolol −15.1±2.4 to −29.5±4.9 mV, P<0.05, n=5; ICI 118551 −9.1±1.7 to −12.1±2.1 mV, P<0.05, n=7).
Figure 4 shows the effects of β-blockers on the HERG peak tail current during a drug wash-in protocol at various drug concentrations. HERG current was elicited from a holding potential of −80 mV by a 4-s depolarising step to 20 mV followed by a repolarising step to −50 mV for 6 s, and the protocol was applied every 15 s. After obtaining the control record, β-blockers were applied to the bath. Application of metoprolol, carvedilol, propranolol and ICI 118551 resulted in a rapid HERG channel blockade to reach a nearly steady-state level within 5 min. The blocking effects were reversed gradually during washout (45–88%). Atenolol at 1 mM did not induce marked reduction of the HERG tail current.
The concentration–response relationship of drug block was assessed by changes in the HERG peak tail current amplitude at 5 min after application of the drug. Currents in the presence of varying concentrations of β-blockers were normalised to the control current amplitude and plotted as a function of drug concentration. The concentration-response curves were obtained by using the Hill equation:
where D is the drug concentration, IC50 is the drug concentration for 50% block, and n is the Hill coefficient. Metoprolol, carvedilol, propranolol and ICI 118551 reduced HERG current amplitude in a concentration-dependent manner but atenolol did not inhibit HERG channels for up to 1 mM (Figure 5). The IC50 value and Hill coefficient are summarised in Table 2.
Figure 5. Concentration–response relationships for peak tail current in HERG wild-type, Y652A and F656C mutant channels. Currents in the presence of varying concentrations of β-blockers were normalised to the control amplitude and plotted as a function of drug concentration: (a) metoprolol, (b) atenolol, (c) carvedilol, (d) propranolol, (e) ICI 118551. Data are mean±s.e.m. Numbers shown near the data points indicate the number of cells tested. The effects of atenolol on Y652A and F656C mutant channels were not tested. Solid lines represent fits with a Hill equation (see text for detail).
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