Blockade of HERG cardiac K+ current by antifungal drug miconazole

Abstract

1. Miconazole, an imidazole antifungal agent, is associated with acquired long QT syndrome and ventricular arrhythmias. Miconazole increases the plasma concentration of QT-prolonging drugs by inhibiting the hepatic cytochrome P450 metabolic pathway, but whether it has direct effects on cardiac ion channels has not been elucidated. 2. To determine the mechanism underlying these clinical findings, we investigated the effect of miconazole on human ether-a-go-go-related gene (HERG) K+ channels. 3. HERG channels were heterologously expressed in human embryonic kidney 293 (HEK293) cells and whole-cell currents were recorded using a patch-clamp technique (23 degrees C). 4. Miconazole inhibited HERG peak tail current in a concentration-dependent manner (0.4-40 microM) with an IC50 of 2.1 microM (n=3-5 cells at each concentration, Hill coefficient 1.2). HERG block was not frequency-dependent. It required channel activation, occurred rapidly, and had very slow dissociation properties. 5. The activation curve was shifted in a negative direction (V(1/2): -9.5+/-2.3 mV in controls and -15.3+/-2.4 mV after 4 microM miconazole, P<0.05, n=6). Miconazole did not change other channel kinetics (activation, deactivation, onset of inactivation, recovery from inactivation, steady-state inactivation). 6. The S6 domain mutation, F656C, abolished the inhibitory action of miconazole on HERG current indicating that miconazole preferentially binds to an aromatic amino-acid residue within the pore-S6 region. 7. Our findings indicate that miconazole causes HERG channel block by binding to a common drug receptor, and this involves preferential binding to activated channels. Thus, miconazole prolongs the QT interval by direct inhibition of HERG channels.

Figure 1

Current–voltage relationship for HERG channels and blockade by miconazole. (a) HERG currents under control conditions and in the presence of 4 μM miconazole recorded using the pulse protocol are shown. (b and c) Normalised (to respective control values) I–V relationships for current measured at the end of depolarising steps (b) and tail currents (c) in the control and the presence of 4 μM miconazole (n=6). Solid lines represent fits to Boltzmann function.

Figure 2

Miconazole inhibits HERG channels. (a) Representative current traces under control conditions and after application of miconazole (40 μM, the 1st, 2nd, 3rd, 4th and 10th current traces are shown). (b) Time course of miconazole-induced HERG tail current inhibition from the same cell shown in (a). (c) Concentration–response relationships for steady-state current amplitude measured at the end of depolarising step to 20 mV and peak tail current (n=3–5 cells at each concentration). Currents in the presence of miconazole were normalised to the control amplitudes and plotted as a function of drug concentration. Data represent the mean±s.e.m. Numbers shown in parentheses indicate the number of cells tested. Solid lines represent fits with Hill equation: I_drug/I_control=1/[1+(D/IC₅₀)ⁿ], where D is the drug concentration, IC₅₀ is the drug concentration for 50% block, and n is the Hill coefficient. (d) Mean relative tail current amplitudes after application of 40 μM miconazole for HERG wild-type (n=3) and F656C mutant (n=7), respectively.

Figure 3

State-dependence of HERG channel block by miconazole. (a) Representative current recordings with a 300-ms long step to 80 mV (protocol 2) compared with a continuous step to 0 mV (protocol 1), see text for details. (b) First pulse current trace in the presence of miconazole (4 μM) with protocol 2. The current decay during the initial step to 0 mV was fitted with a single exponential function, which was extrapolated to the end of pulse protocol. The current amplitude at the beginning of the second step to 0 mV exceeded the extrapolated predicted current. (c and d) The initial current amplitude was not reduced by 4 μM miconazole but was reduced by 12 μM miconazole.

Figure 4

Inhibition of HERG tail current by miconazole is not frequency-dependent. After the cell was held at −80 mV for 3 min during exposure to 4 μM miconazole, 30 repetitive pulses (see inset) were applied at 1 and 0.2 Hz. HERG current amplitude measured as peak value during step to 20 mV for each pulse was normalised by the control peak current amplitude, and then plotted against the pulse number. For control conditions, there was little effect of the pulse train applied at 0.2 or 1 Hz (n=5), whereas in the presence of miconazole, HERG current was reduced similarly at 0.2 and 1.0 Hz pulse frequencies. Solid lines represent fits with double exponential function: A_f exp(−t/τ_f)+A_s exp(−t/τ_s)+base, where t is pulse number; τ_f and τ_s are time constants of fast and slow components; A_f and A_s are fractional amplitudes of fast and slow components. Fitting parameters were τ_f 1.16±0.12, τ_s 75.3±37.1 at 1 Hz (n=5) and τ_f 1.24±0.15, τ_s 64.7±17.8 at 0.2 Hz (n=5).

Figure 5

Effects of miconazole on activation time course and deactivation kinetics. (a) Representative current tracings for HERG current activation before and after application of 4 μM miconazole. Pulse protocol is shown in the inset. (b) The peak tail current after a repolarising step to –50 mV was plotted as a function of the test pulse duration (ΔT). Solid lines represent fits with a single exponential function. Data are expressed as mean±s.e.m. (n=8). (c) Representative current tracing for HERG current deactivation and pulse protocol (bottom inset). Deactivation time constants were obtained by fit with double exponential function to the decay phase of tail current. (d) Time constants for the deactivation are plotted against the membrane potential. Data are expressed as mean±s.e.m. (n=5).

Figure 6

Effects of miconazole on inactivation kinetics. (a) Representative current tracing for HERG current recovery from inactivation elicited by the protocol shown below. Region of interest is magnified. After fitting to tail current with double exponential function, the time constant for the rising phase was used to evaluate the recovery. (b) Representative current tracing for HERG current onset of inactivation by using the protocol shown below. The time constants for the onset of inactivation were obtained by fitting exponential function to the decaying current traces during the third pulse of the protocol. (c) The time constants for the recovery and onset of inactivation are plotted against the membrane potential. Data are expressed as mean±s.e.m. (n=5, ^*P<0.05, control vs 4 μM miconazole). (d) Representative current tracing for steady-state inactivation by using a double-pulse protocol with varying interpulse repolarisation levels from −100 to 20 mV (bottom inset). (e) Normalised steady-state inactivation curves for control and after 4 μM miconazole application. Data are expressed as mean±s.e.m. (n=6). Solid lines represent fits with Boltzmann function.