Pentamidine reduces hERG expression to prolong the QT interval

Abstract

Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC50 of 252 microM. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 microM, with a slight prolongation of the action potential duration at this concentration. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (approximately 1 microM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 microM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-microM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.

Figure 1

Pentamidine block of the hERG current in stable hERG-HEK cells. (a) Representative current traces before and after administration of pentamidine at various concentrations. The voltage protocol used to elicit the hERG current is shown in the upper panel. (b) Concentration–response curve of the pentamidine effect. Averages of 4–5 experiments were fitted with a Hill equation and an IC₅₀ of 252 μM was obtained.

Figure 2

Voltage-dependent blocking of pentamidine on the hERG channel. (a) Typical current traces at different voltages in the absence (Ctrl) and presence of 250-μM pentamidine (Pent). Cells were held at −80 mV, depolarized to a series of steps ranging from −70 to +40 mV for 1 s, and followed by a repolarization pulse at −70 mV to elicit the tail current. (b) Current–voltage (I–V) relationship of the hERG channel. Developing currents measured at the end of each depolarization pulse were summarized from five experiments. Open and closed squares indicate control and 250 μM pentamidine groups, respectively. (c) Effect of pentamidine on the voltage dependence of steady-state activation (square symbols). Averaged data from five experiments were fitted with a Boltzmann function, resulting in a half-activation voltage and a slope factor of −32.0 and 6.0 mV, respectively, for the control (open squares), and −36.6 and 5.3 mV, respectively, for pentamidine (closed squares). (d) Voltage-dependent block by pentamidine was computed from the tail currents in the presence of drug normalized to the control (I_Pent−I_Ctrl⁻¹). A single exponential function was applied to fit the averaged data from five observations.

Figure 3

Blocking mechanism of pentamidine. (a) Pentamidine block of hERG during continuous stimulation (open squares) and after holding the cell at −80 mV for 5 min (open circles) to keep the channel at closed state. A 5-min period was sufficient for a steady-state block during normal perfusion and stimulation when pentamidine (250 μM) was administered. Currents were elicited by a voltage protocol used for the potency determination (Figure 1) as shown in the inset. Tail current amplitude was measured and plotted. Note that the first stimulation after the 5-min pause elicited a percentage of current comparable to that at the steady-state block level and no further increase of the block was observed following consecutive stimuli. (b) Current traces at 0 and +20 mV in the presence and absence of 250-μM pentamidine. Voltage protocol was shown in the inset. No apparent time-dependent development of blocking was observed in the depolarization-activated currents at both potentials. (c) Pentamidine block does not require channel activation. Using a voltage protocol shown at the top, hERG current was rapidly activated from a holding potential of −80 mV by a 5-ms depolarizing step to 100 mV, followed by a step to 0 mV for 100 ms. The first stimulation after 5-min wash-in of 250 μM pentamidine elicited a significantly reduced (∼50%) current which was almost in parallel to the control. Similar results were observed in four experiments. (d) Pentamidine block during a strong membrane depolarization. Currents were recorded from a representative cell before and after application of 250-μM pentamidine. Voltage protocol shown in the upper panel included a first depolarization at 0 mV for 1 s, then at +70 mV for 2 s, and finally at 0 mV again for 1 s. Percent block at the end of each step was computed.

Figure 4

Effects of pentamidine on the APs (a) and L-type calcium channel current (b) in freshly isolated guinea-pig ventricular myocytes. APs were recorded by using perforated-patch technique and elicited by suprathreshold stimulations under current-clamping mode. L-type calcium currents were elicited by a depolarization to +10 mV from a prepulse at −50 mV. Current traces shown in upper panel b were from a representative experiment, and the superimposed recordings, from bottom up, represent control, 10, 30, 100 and 300 μM pentamidine, respectively. In the time course of the peak current amplitude (lower panel b), a linear fit to the control values was extrapolated throughout the experiment, which indicated no noticeable effect of pentamidine at the test concentrations.

Figure 5

Pentamidine inhibits the surface membrane expression of the hERG channel. (a) Decreased hERG current as a result of pentamidine treatment. The hERG-HEK cells were incubated for 48 h in culture media containing 1- or 10-μM pentamidine or its vehicle before the patch-clamp measurement. Current traces were elicited by the same voltage protocol as shown in Figure 1. Current densities were summarized from the three groups (n=26, 32 and 23 for control, 1 and 10 μM pentamidine, respectively). ^***P<0.001. (b) Decrease of hERG polypeptide in the pentamidine-treated cells detected by Western blot analysis. Whole-cell lysates were prepared and electrophoresed on a 4–12% bis-acrylamide gel, and subsequently transferred to nitrocellulose. The membrane was probed with a hERG antibody (upper gel), stripped and reprobed with an antibody against GAPDH (lower gel). The hERG band (∼155 kb, upper gel) densities were measured, normalized against the corresponding GAPDH levels, and are summarized in lower panel b.

Figure 6

Pentamidine exposure to hERG-HEK cells reduces surface membrane expression of the hERG channel. Laser-scanning confocal microscopy optical sections of control hERG-HEK cells (a) and cells in the presence of 1 μM pentamidine (b). The hERG polypeptide was recognized using an indirect fluorescence method and pseudocolored red; DNA was labeled with an intercalating dye and pseudocolored blue. Arrowheads in (a) and (b) are the bounds of the line profile for fluorescence intensities. The line begins with the left arrowhead and stops at the right arrowhead. Fluorescence intensities for both hERG polypeptide (single arrow) and DNA (double arrow) are graphically represented on the histogram plot. A single arrow marks a region of cell membrane, with a double arrow marking the nucleus. Changes in both hERG and DNA fluorescence signals can be appreciated along the line profiles. Scale bar=10 μm.