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From the Department of Anesthesiology, *Renmin Hospital of Wuhan University, and Tongji Hospital of Huazhong University of Science and Technology, Wuhan, China.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS: Wild type and mutant HERG channels were heterologously expressed in human embryonic kidney 293 cells, and the current was recorded by using whole cell patch clamp technique (22–24°C).
RESULTS: HERG tail currents following test pulses to 50 mV were inhibited by droperidol with an IC50 of 77.3 ± 9.6 nM (n = 8). The onset of block was fast and inhibition was completely reversible upon washout. Droperidol affected HERG channels mainly in their open and inactivated states. The effects were use-dependent with a stronger steady-state level of block at higher frequencies. The activation curve was slightly shifted towards more negative potentials (P < 0.05, n = 8) and the time course of inactivation was significantly decreased (P < 0.05, n = 8) by 100 nM droperidol. But there was no relevant effect on HERG channel deactivation. The potency for block of HERG channels by droperidol was significantly decreased with mutation of Phe-656 to Thr or mutation of Ser-631 to Ala, respectively. However, mutation of Phe-656 to Met or the double mutation F656M/S631A had no effect on channel sensitivity to block by droperidol.
CONCLUSIONS: Droperidol potently inhibits transfected HERG channels and this is the probable mechanism for QT prolongation. Channel blockade shows greatest affinity for the open and inactivated state. Aromatic residue at position 656 may participate in droperidol binding, and inactivation gating can induce a conformational state that optimizes droperidol binding to the channel.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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IKr was first identified in guinea pig ventricular and atrial cells,4,5 and the properties of IKr have subsequently been characterized in a wide variety of other mammalian cardiac myocytes, including from human heart.6 This outward current is crucial in determining the rapid repolarization of phase 3 of the cardiac action potential (and thus the QT interval). It has been shown that the underlying gene of the IKr current is the human ether-a-go-go-related gene (HERG).7,8 Mutations in the HERG gene cause an inherited disease known as Long QT Syndrome, a disorder that predisposes individuals to life-threatening arrhythmias. However, in clinical practice, a large number of drug-induced QT prolongation and TdP are caused by direct blockage of HERG channels.9
Droperidol has also been confirmed to be a potent blocker of heterologously expressed HERG channels in addition to its inhibition on natural Ikr.3,10 However, biophysical properties and molecular determinants for droperidol’s block of the HERG channel have not been adequately studied. To more accurately characterize the drug-channel interaction, we conducted the present study to explore in detail how droperidol affects HERG channel function. Because both an intact HERG C-type inactivation gating and the S6 aromatic amino-acid residues (Y652 and particularly F656) are important for high-affinity binding of chemically and therapeutically diverse drugs,11,12 we therefore further evaluated the changes to droperidol sensitivity by applying site-directed mutagenesis at the position of S631 and F656. Our results imply that droperidol can interact with both the open and inactivated states of the HERG channel. Aromatic residue at position 656 is required for potent HERG channel block by droperidol, whereas inactivation gating can induce a conformational state that optimizes droperidol binding to the channel.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Electrophysiological Recordings
HERG channel currents were recorded using the whole cell patch clamp technique. The pipette solution contained 135 mM KCl, 5 mM EGTA, 1 mM MgCl2, 10 mM HEPES, and was adjusted to pH 7.2 with 1 M KOH. The bath solution contained 130 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, 1 mM MgCl2, 2 mM CaCl2 and was adjusted to pH 7.4 with 2 M NaOH. Aliquots of cells were allowed to settle on the bottom of <0.5 mL cell bath mounted on an inverted microscope (Nikon). Solution exchanges in the cell bath were completed within 10 s. Micropipettes were pulled from thin-walled borosilicate glass capillary tubes (World Precision Instruments, Sarasota, FL) on a programmable puller (Sutter Instruments, Novato, CA). The pipette had inner diameters of 1–1.5 µm and had resistances of 2–4 M[Omega] when filled with the internal pipette solution.
Data acquisition and generation of voltage-clamp pulse protocols were performed with a Digidata 1322A interface (Molecular Devices Corp, Sunnyvale, CA) controlled by pCLAMP 9 software (Axon Instruments, Foster City, CA). Membrane currents were recorded with an Axopatch 200B amplifier (Axon Instruments). Current signals were filtered at 2–5 kHz and sampled at 10–20 kHz. After obtaining whole-cell access, series resistance was compensated to minimize the duration of the capacitive transient. Typically, 80% series resistance (Rs) compensation was used and leak subtraction was not used. All experiments were performed at room temperature (22–24°C). Specific voltage-clamp protocols for each experiment are described in the Results section.
Chemicals
Droperidol was purchased from the Sigma Aldrich (D-1414) and was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (100 mM). Final drug concentrations were prepared by diluting stock solution with bath solution. The highest concentration of DMSO used in this study was 0.003%. In preliminary experiments, HERG tail current amplitudes expressed in HEK293 cells were not significantly changed after 3 min of DMSO application at 0.1% (data not shown).
Data Analysis
Curve fitting was done using multiple nonlinear least-squares regression analysis (Origin, Microcal Software; Clampfit, Axon Instruments). The concentration– effect relationships were fitted to the Hill equation {Idrug/Icontrol = 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 to the results. The voltage-dependence of current activation and inactivation was determined by fitting the values of the normalized tail currents to a Boltzmann function as previously described7: I = Imax x [1 + exp (V1/2 – V)/k]–1, where Imax is maximum amplitude, V1/2 and k are half-maximal voltage and the slope factor, respectively.
Data are expressed as mean ± sem. The "n values" in the text indicate the number of cells used in whole cell patch clamp experiments. After confirmation of normal distribution of the data by using the Shapiro-Wilk test, statistical significance was assessed by paired or unpaired t-test, as appropriate. A P value <0.05 was considered to be statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the time course of onset and washout of block by droperidol in further detail, we studied the effects of 1000 nM droperidol on the HERG current during a drug wash-in and washout protocol (Figs. 1C and D). The voltage protocol was repeated at 15-s intervals until a steady-state was reached. After a control period of 4 min, 1000 nM droperidol was applied by perfusing the bath. The steady-state block was rapidly obtained after 1 min of droperidol application, and the inhibitory effect was completely reversible within 3 min after drug washout. The normalized tail current amplitude plotted versus time is shown in Figure 1E (n = 8 cells).
Droperidol Blocks HERG Potassium Channel in the Open and Inactivated State
To determine whether the channel is blocked in the closed, open, or inactivated state, two different approaches were chosen to investigate the state-dependent block of HERG channels by droperidol. In protocol 1, from a holding potential of –80 mV, the cell was first depolarized to 80 mV for 100 ms to maximally activate channels from the closed state, and then the cell was repolarized to 0 mV for 8.5 s to allow for maximal channel recovery from inactivated to the open state. After having obtained the control measurement (Fig. 2A upper panel), 100 nM droperidol was washed-in for 3 min while holding all channels in the closed state at –80 mV without pulsing. With the first depolarization in the presence of droperidol, the initial HERG current amplitude was unchanged compared with the control current, indicating minimal closed state block. The current amplitude then declined during the maintained depolarization at 0 mV to reach a steady level of drug block (Fig. 2A lower panel). Apparently, there is mainly a block of open or inactivated channels with no marked inhibition of closed channels. Subsequent depolarizing pulses at 6-s intervals showed no time-dependent component of block, thus indicating that droperidol had not dissociated appreciably from the channel within this time frame.
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In protocol 2, HERG channels were depolarized to 80 mV for 100 ms and then repolarized to 0 mV for 1 s, followed by a 1.5 s intervening step to 80 mV, before returning to 0 mV for 6 s. This approach of using steps to very positive potentials was designed to determine whether maximal drug block occurred when channels were in the inactivated states. Figure 2B (upper panel) shows example control records with this protocol. There was less current during the 1.5 s long step to 80 mV because of increased channel inactivation. The recording of control measurements was followed by application of 100 nM droperidol to the HERG HEK cell for a period of 3 min, during which the cell was held at –80 mV without pulsing. As shown in Figure 2B (lower panel, dashed frame), there was a decline in HERG current during the second depolarization to 80 mV. In eight cells studied with this protocol, the currents remaining at the beginning of the second 80 mV step were 30.7 ± 3.6% of the initial value, and declined to 20.3 ± 2.1% at the end of the second 80 mV step. The difference was statistically significant by the paired t-test (P < 0.05). These results suggest that blockade by droperidol also occurs when the HERG channels are predominantly in the inactivated state.
Frequency Dependence of HERG Channel Block by Droperidol
The frequency dependence of HERG current block was investigated by applying 30 repetitive pulses (Fig. 3) at intervals of 1 and 15 s. HERG current was first rapidly activated by a 100 ms step to 80 mV, which was followed by a 400 ms step to 20 mV. HERG current amplitude measured as peak value during step to 20 mV for each pulse was normalized by the control peak current amplitude, and then plotted versus the pulse number. After control, the cells were exposed to 100 nM droperidol for 3 min at –80 mV without pulsing. Only one cell was used at each frequency. For control conditions, there was little effect of the pulse train applied at 1 or 15 s intervals (n = 8, P > 0.05, unpaired t-test), whereas 100 nM droperidol reduced current amplitude to 43.8 ± 4.5% (n = 8) of control after 30 pulses at 1 s interval and 54.2 ± 5.5%(n = 8) of control after the same number of pulses at 15 s interval, respectively (P < 0.05, unpaired t-test). Therefore, droperidol block was use-dependent with a stronger steady-state level of block at higher frequencies.
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Effects of Droperidol on HERG Activation
To characterize the effect of droperidol on HERG activation, HERG currents elicited by the voltage protocol were compared before and after superfusion of 100 nM droperidol. Figure 4A shows HERG currents under control condition and in the presence of 100 nM droperidol recorded from a holding potential of –80 mV by 4 s long depolarizing steps to between –70 and 70 mV applied in 10 mV increments at 15 s intervals. Tail current was recorded after the repolarizing step to –50 mV. Figure 4B shows averaged current–voltage (I–V) relations for HERG current measured at the end of depolarizing steps (Fig. 4B upper panel) and for the subsequent tail current peak amplitude(Fig. 4B lower panel) for control conditions and with 100 nM droperidol in the same cells. Under control conditions, HERG currents activated at voltages more than –50 mV, reached a peak at 10 mV, and then decreased at more positive potentials due to rapid inactivation,7,15 giving the I–V relationship and its typical bell-shaped appearance. The peak tail current, measured during the repolarizing second step of the voltage protocol, increased with voltage steps from –40 to 20 mV and then plateaued for test pulse potentials positive to 20 mV. At a concentration of 100 nM, droperidol’s block of HERG current was significant at all voltages between –20 and 70 mV. HERG currents at the end of the test pulse to 10 mV were reduced by 39.3 ± 7.9% (n = 8), and peak tail currents were blocked by 41.4 ± 4.4% (n = 8). Figure 4C displays activating and peak tail currents normalized to their respective peak values as a function of the test pulse potential, resulting in activation curves. Data from normalized tail currents were fitted with a Boltzmann function. In this protocol, the half-activation voltage was significantly shifted in a negative direction from –11.4 ± 0.2 mV (n = 8) in controls to –17.6 ± 0.4 mV (n = 8) after droperidol (P < 0.05, paired t-test). However, there was no significant change in the slope factor after droperidol (P > 0.05, n = 8, paired t-test).
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Effects of Droperidol on HERG Inactivation
To measure the time constant of inactivation directly, we used a three-pulse protocol.16 In this protocol, HERG current was activated and inactivated by a 500-ms-long depolarizing step to 60 mV from a holding potential of –80 mV. The cell was then repolarized to –100 mV for 10 ms to allow for recovery from inactivation without significant deactivation of HERG current. Then, channels that recovered were forced to reinactivate at potentials between –20 and 120 mV. The currents elicited by the test steps were of large amplitude and were rapidly inactivated. Control inactivating currents (Fig. 5A, left panel) and currents under 100 nM droperidol (Fig. 5A, right panel) were fitted with single exponential functions to obtain time constants (Fig. 5B). The two graphs describing the time constants under control conditions and with droperidol were significantly different (P < 0.05, n = 8, paired t-test).
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Steady-state inactivation was measured using a double-pulse protocol with varying interpulse repolarization levels. Channels were inactivated at a holding potential of 60 mV, before being recovered from inactivation at various potentials from –100 to 20 mV for 20 ms. Finally, the resulting peak outward currents at constant 60 mV as a measure for steady-state inactivation were recorded. Data from eight cells under control and 100 nM droperidol were normalized to their respective maximal value and plotted versus the prepulse voltage, giving the steady-state inactivation curve (Figs. 5C and D). The half inactivation voltage and slope factor obtained by fitting with Boltzmann equation were –79.6 ± 2.5 mV and 21.2 ± 1.5 in controls (n = 8), and –78.4 ± 2.6 mV and 21.6 ± 1.2 after 100 nM droperidol (n = 8), respectively. Parameters were not statistically different (P > 0.05, n = 8, paired t-test).
Effects of Droperidol on HERG Deactivation and Recovery from Inactivation
The time constant of deactivation was measured using a double-pulse protocol, in which the cell was depolarized to 60 mV for 400 ms to activate and inactivate HERG channels, and then was repolarized to voltages between –10 and –150 mV to give a tail current. The decay phase of the tail current represents HERG deactivation and the rising phase of the tail current represents the rapid recovery of HERG channels from inactivated to open states. The time constants of deactivation and recovery from inactivation were obtained by fitting with double exponential function. There were no significant differences before and after 100 nM droperidol (P > 0.05, n = 8, data not shown).
Molecular Determinants for Droperidol Binding to HERG
To gain additional information on the molecular basis of droperidol binding to HERG channels, we investigated the effects of droperidol on HERG F656T, F656M, S631A, and F656M/S631A channels. F656T and F656M in the S6 domain maintain the kinetics and voltage dependence of gating similar to WT HERG.17 S631A in the P-loop of the outer pore causes a +100 mV shift in the voltage dependence of inactivation, but no shift in activation compared with WT channels.18 The affinity of droperidol to HERG WT and mutant channels was compared by measuring the effects of droperidol at 100 nM. Voltage protocols were applied as described in Figure 1 to record peak tail currents.
Examples of steady-state block of WT and four mutant HERG channels are shown in Figure 6A. For each channel, the changes in drug sensitivity were calculated by dividing the current in presence of droperidol by the value of current before drug administration. As can be seen in Figure 6B, in WT HERG channels, the tail current peak amplitudes were reduced to 44.8 ± 3.0% (n = 6) of the control current amplitude. The inhibitory effect of droperidol on HERG current was almost completely abolished in the F656T mutant channels (96.7 ± 0.8%, P < 0.01 vs. WT, n = 6, unpaired t-test); however, the blockade of the F656M mutant was not significantly different compared with that of the WT channel (45.5 ± 2.0%, P > 0.05, n = 6, unpaired t-test). We also determined the sensitivity of inactivation deficient mutant S631A to block by droperidol. When using 100 nM droperidol, the mutation produced a modest attenuation of blockade compared with the WT channel (79.7 ± 2.4%, P < 0.01, n = 6, unpaired t-test). To further address whether the F656M mutation alters the sensitivity for droperidol in a channel that does not inactivate (S631A), the double mutation F656M/S631A was made. The mean relative peak amplitude of the tail current measured after droperidol application yielded 46.3 ± 2.9% (n = 6) of the control current amplitude, which was not significantly different than is observed for the individual mutations F656M or for WT HERG.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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We showed that HERG channel block occurred rapidly to reach a steady-state level within 1–2 min. It is interesting to correlate these findings with the data from previous clinical observations. In a study of adult patients with postoperative nausea and vomiting, Charbit et al. found that QT prolongation occurred at 2 min after administration of droperidol (0.75 mg IV).22 It has also been reported that neuroleptanalgesic doses of droperidol (100 µg/kg) led to significant QT prolongation within 5 min of bolus administration in children undergoing cardiac surgery.23 On the basis of these studies, we suggested that the fast onset of HERG block by droperidol might contribute to the prompt development of QT prolongation seen clinically with administration of the drug.
The present study was designed to analyze the biophysical mechanism of HERG channel block by droperidol to better understand the possible proarrhythmic properties of this drug. One important finding of this study was that droperidol blocked HERG channels with high affinity in the open and inactivated state, whereas closed HERG channels were not significantly affected by the drug molecule. First, droperidol block proceeds during, but not before, long depolarizing pulses to 0 mV, during which maximal conductance is usually observed. Second, interruption of prolonged steps to 0 mV by the application of short pulses (1.5 s) to 80 mV that cause instant and complete inactivation did not hinder the blocking process. Thus, the binding site is only accessible for droperidol when the channel is in the activated state.
We observed that channel inhibition by droperidol was frequency-dependent, with the block being stronger at higher stimulation frequencies. This frequency-dependence can be accounted for by a fast unbinding of the drug from the channel. With relative fast drug dissociation, the longer recovery interval between pulses at slower rates caused appreciable additional recovery over the time scale studied in our experiments. The fast current recovery after drug washout (Fig. 1D) also supports the notion of fast channel dissociation of droperidol. Our findings contrast with those of Schwoerer et al., who used a similar treatment protocol with Xenopus oocytes expressed HERG channels and showed no significant effect of droperidol on HERG frequency dependent block.10 These differences may be attributed in part to differences in the expression systems used (Xenopus oocytes versus HEK293 cells), experimental conditions (difference in pulse frequency), and channel kinetics.
To further characterize the block of HERG by droperidol, we assessed the effects of droperidol on the kinetics of activation, inactivation, and deactivation of the HERG channel. We found that in the presence of droperidol, a slight shift of the mean half-maximal activation voltage V1/2 by 6 mV towards more negative potentials was observed, which was significant. However, this small effect is not expected to contribute considerably to the effects of droperidol in vivo. We found that droperidol accelerated the inactivation time course of the HERG channels, which also provided suggestive evidence for the inactivation block of the HERG channels by droperidol. As with most drugs, droperidol had no relevant effect on HERG channel deactivation.
To locate the binding site of droperidol, we used several mutants at the position of F656 and S631, that are critical for HERG block by a large number of long QT-inducing drugs.11,12 We found that mutation of Phe-656 to the hydrophobic residue Met (F656M) retained normal sensitivity to droperidol, whereas mutation of Phe-656 to polar residue Thr (F656T) was relatively insensitive. This result supports the theory that the important physicochemical feature of Phe-656 is hydrophobic volume, not aromaticity per se. Moreover, the point mutant in the P-loop region (S631A), which removes the rapid channel inactivation, also reduced sensitivity to inhibition by droperidol. To further identify whether inactivation or Phe-656 is a critical determinant of the high-affinity binding of droperidol, a double mutation was generated, including both S631A and F656M. Since F656M had little effect in droperidol binding in our study, the double mutation F656M/S631A could help to elucidate the role of S631 in the drug channel interaction. We reasoned that if inactivation is necessary for high-affinity droperidol binding, the sensitivity of the double mutation to droperidol should be generally similar to that seen with S631A, no matter what amino acid is present at the Phe-656 position. However, our data showed that the sensitivity of the double mutation to droperidol was not significantly different than is observed for the individual WT HERG or F656M mutation. Therefore, the affinity of the channel to droperidol produced by the F656M mutation was unrelated to the disruption of inactivation. Together, these data suggest that the S6 residue F656 is an important structural determinant of droperidol block, and inactivation-associated allosteric changes of Phe-656 facilitate droperidol binding to HERG. However, our results cannot exclude the possibility of inactivation gating associated reorientation of F652 that comprise the droperidol binding site as suggested by Schwoerer et al.10 Future studies might include using more site-directed mutagenesis to target potential binding sites of droperidol in the HERG channels.
Block of HERG K channels is a risk factor in drug-induced TdP, and probably the predominant risk factor, but it requires a combination of factors to trigger the event.24 Our study provided a direct cellular mechanism for droperidol-induced QT interval prolongation and cardiac arrhythmias. This information is important for the scientists developing medications, and for the physicians prescribing them. Because clinical factors and concomitant medications play a crucial role in arrhythmic events, HERG channel blockade might be particularly important when droperidol is used in patients with congenital long QT syndrome, in patients with electrolyte abnormalities such as hypokalemia, or in patients receiving additional drugs know as inhibitors of HERG channel. It is also important to note that, on the basis of inactivation facilitated HERG-channel blockade by droperidol, blockade of Ikr would be more pronounced during cardiac infarction or myocardial ischemia, because membrane depolarization occurs under these situations.
In conclusion, droperidol potently blocks HERG K+ channels expressed in HEK 293 cells in a concentration-, state- and frequency-dependent manner. Aromatic residue at position 656 may participate in droperidol binding, and inactivation gating can induce a conformational state that optimizes droperidol binding to the channel. This finding provides a molecular mechanism for the previously reported QT interval prolongation under clinical administration of droperidol.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supported in part by NSFC Grant 30700789.
Address correspondence and reprint request to Dr. Tao Luo, Department of Anesthesiology, Renmin Hospital of Wuhan University, 99 Ziyang Road, Wuhan 430060, China. Address e-mail to luotao_wh{at}yahoo.com.
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REFERENCES |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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