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Meeting Abstracts  |   May 2007
Molecular Interaction of Droperidol with Human Ether-a-go-go  -related Gene Channels: Prolongation of Action Potential Duration without Inducing Early Afterdepolarization
Author Affiliations & Notes
  • Alexander P. Schwoerer, M.D.
    *
  • Carmen Blütner, B.Sc.
  • Sven Brandt, M.D.
  • Stephan Binder, M.Sc.
    §
  • Cornelia C. Siebrands, Ph.D.
  • Heimo Ehmke, M.D.
    #
  • Patrick Friederich, M.D.
    **
  • * Postdoctoral Fellow, # Professor and Chairman, Department of Vegetative Physiology and Pathophysiology, † Graduate Doctoral Student, ‡ Resident, § Diploma Student, ** Privatdozent, Department of Anesthesiology, ∥ Postdoctoral Fellow, Department of Anesthesiology and Institute of Neural Signal Transduction, University Medical Center, Hamburg-Eppendorf, Germany.
Article Information
Meeting Abstracts   |   May 2007
Molecular Interaction of Droperidol with Human Ether-a-go-go  -related Gene Channels: Prolongation of Action Potential Duration without Inducing Early Afterdepolarization
Anesthesiology 5 2007, Vol.106, 967-976. doi:10.1097/01.anes.0000265156.09438.56
Anesthesiology 5 2007, Vol.106, 967-976. doi:10.1097/01.anes.0000265156.09438.56
DROPERIDOL is a highly potent butyrophenone that has been commonly used for more than 30 yr in the therapy of postoperative nausea and vomiting.1,2 High doses of droperidol (0.25 mg/kg) given for the induction and maintenance of anesthesia have been reported to prolong the QT interval in perioperative patients.3 After several cases of excessive QT-prolongation leading to fatal arrhythmias associated with the use of droperidol at high doses, the US Food and Drug Administration released a “black box” warning in 2001.1As a consequence, the clinical use of droperidol in postoperative nausea and vomiting therapy has decreased dramatically.2 However, not a single case of cardiac arrhythmia has been reported after application of droperidol at antiemetic doses (0.625–1.25 mg).2,4 Furthermore, droperidol given at antiemetic doses does not seem to be associated with a clinically significant prolongation of the QT interval.5 Also, the cardiotoxic potential of droperidol at antiemetic doses has not been addressed in controlled clinical trials.2,4,6,7 The cardiac safety of droperidol in postoperative nausea and vomiting therapy, therefore, remains a matter of debate.
Prolongation of the QT interval reflects an increase of action potential duration (APD) in ventricular cardiomyocytes. Contradicting results have been obtained in animal studies regarding the effects of droperidol on APD and its potential to induce early afterdepolarizations (EADs).8–12 Some authors demonstrated action potential (AP) shortening,8,9 whereas others reported AP prolongation11,12 or no effect on APD.10 Drug-induced prolongation of cellular APD and induction of torsades de pointes ventricular arrhythmia is often caused by high-affinity block of the delayed rectifying K+current IKr.13 The channel underlying IKris encoded by the human ether-a-go-go  -related gene (HERG).14,15 High-affinity block of HERG channels is transmitted by two aromatic amino acid residues in the S6 helix (Tyr652 and Phe656) and by several residues located at the base of the pore helix (Thr623, Ser624, Val625).16–20 Based on the concentration of half-maximal inhibition (IC50) value for inhibition of HERG channels, droperidol may be regarded as a high-affinity blocker.12 Currently, however, it is unknown whether the amino acid residues typically mediating high-affinity block are involved in the interaction of droperidol with HERG channels. Furthermore, it is unknown whether droperidol interacts with these channels in a way mechanistically similar to high-affinity blockers such as dofetilide and E-4031.19,21 
Therefore, despite the “black box” warning of the US Food and Drug Administration on the cardiac safety of droperidol, several issues of the cardiotoxic profile of droperidol remain unresolved. The molecular mode of interaction of the drug with HERG channels has not been defined. Furthermore, it is unclear whether droperidol at antiemetic concentrations induces AP prolongation and early afterdepolarizations in ventricular cardiomyocytes. The aim of our study, therefore, was to characterize the molecular mode of interaction of droperidol with HERG channels. In addition, it was intended to suggest a possible explanation why droperidol may not induce EADs despite its interaction with HERG channels.
Materials and Methods
All animal experiments were conducted in accordance with institutional guidelines and approved by local authorities (Ministry of Science and Health, Hamburg, Germany).
Solutions and Chemicals
Ringer's solution contained 75 mm NaCl, 2 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 5 mm Na-pyruvate, and 5 mm HEPES, titrated to pH 7.5 with NaOH. Two-electrode voltage clamp experiments were performed using different extracellular solutions: HERG and HERG mutants were investigated using NaCl-79 solution, which contained 79.5 mm NaCl, 2 mm KCl, 2 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES, pH adjusted to 7.5 with NaOH. NaCl-95 solution used for measurement of IKscontained 95 mm NaCl, 2 mm KCl, 1 mm MgCl2, 1 mm CaCl2, and 10 mm HEPES, titrated to pH 7.40 with tris-(hydroxymethyl)-aminomethane. Cardioplegic solution contained 15 mm NaCl, 9 mm KCl, 4 mm MgCl, 0.33 mm NaH2PO4, 0.015 mm CaCl2, 10 mm glucose, and 238 mm mannitol, titrated to pH 7.40 with NaOH. For patch clamp experiments, myocytes were bathed in modified Tyrode solution containing 138 mm NaCl, 4 mm KCl, 1 mm MgCl2, 0.33 mm NaH2PO4, 2 mm CaCl2, 10 mm glucose, and 10 mm HEPES, titrated to pH 7.30 with NaOH. The pipette solution contained 120 mm K-glutamate, 10 mm KCl, 2 mm MgCl2, 10 mm EGTA, 10 mm HEPES, and 2 mm Na2-ATP, titrated to pH 7.20 with KOH. Droperidol (Sigma, Deissenhofen, Germany) was dissolved in dimethyl sulfoxide. The maximal concentration of dimethyl sulfoxide in the respective extracellular solution was 1%, whereas the concentration used for kinetic analysis was less than 0.1%. At these concentrations, dimethyl sulfoxide did not by itself inhibit currents (data not shown), and its effect on HERG channel gating is negligible.22 
Molecular Biology, cRNA Preparation
The HERG mutants T623A, S624A, V625A, Y652A, and F656A were created by site directed mutagenesis.23 All constructs were cloned in the pGEM expression vector for complementary RNA (cRNA) synthesis. The cRNA was synthesized in vitro  with the mMESSAGE mMACHINE Kit (Ambion, Austin, TX) according to the manufacturer's protocol. The cRNA was purified with a phenol/chloroform extraction. The integrity of the cRNA was analyzed in a denaturating gel. The concentration was determined with the RiboGreen method (RiboGreen RNA Quantification Reagent; Molecular Probes, Eugene, OR). For preparation of oocytes, female Xenopus laevis  frogs (n = 37) were anesthetized with tricaine solution, and ovarian lobes were surgically removed. Oocytes were isolated by enzymatic digestion using collagenase A (Roche, Mannheim, Germany; Serva, Heidelberg, Germany) for 2–4 h. Enzymatic digestion was stopped by incubation in Ringer's or NaCl-95 solution. Defolliculated oocytes (stages V and VI) were injected with cRNA of HERG, HERG mutants, or KCNQ1/KCNE1 (in a ratio of 1:1).
Two-electrode Voltage Clamp
Whole cell currents were measured 2–7 days after cRNA injection with the two-electrode voltage clamp technique using an Oocyte-Clamp OC-725C amplifier (Warner Instrument Corporation, Hamden, CT) or a TurboTec 05 amplifier (npi electronic, Tamm, Germany), both controlled by Pulse software (HEKA Elektronik, Lambrecht, Germany). Sharp electrodes were pulled from borosilicate glass capillary tube (World Precision Instruments, Sarasota, FL or Clark Electromedical Instruments, Reading, United Kingdom) and filled with 3 m KCl and 3% agar. Whole cell currents were measured at room temperature (21°–24°C) while oocytes were superfused at a constant rate.
Isolation of Guinea Pig Ventricular Myocytes and Patch Clamp Experiments
For isolation of cardiomyocytes, male Dunkin-Hartley guinea pigs (n = 12; 350–450 g) were deeply anesthetized by thiopental-Na+(200 mg/kg body weight), and hearts were excised and quickly placed into cold (4°C) cardioplegic solution. Myocytes of the center part of the left ventricular free wall were enzymatically isolated as previously described using a Langendorff apparatus.24,25 Single myocytes were stored at room temperature in Ca2+free modified Tyrode solution. Only single rod-shaped cells with clear cross-striations and no spontaneous contractions were used for experiments within 12 h after isolation. The ruptured-patch whole cell configuration was used as previously described26 using an EPC-9 amplifier controlled by the Pulse software. Membrane voltages were recorded in the zero current clamp mode. Pipette potentials were corrected online for liquid junction potentials. APs were elicited at 0.2 Hz by depolarizing current injections of 5 or 10 ms in duration. All experiments were performed at room temperature (21°–24°C) while cells were superfused at a constant rate.
Data Analysis
Data were analyzed using PulseFit software (HEKA Elektronik), Igor (WaveMetrics, Lake Oswego, OR), and KaleidaGraph software (Synergy Software, Reading, PA). The normalized tail currents during the activation protocol were fitted by a modified Boltzmann equation: I = Imax/[1 + exp((V0.5− Vm)/k)], where V0.5is the membrane potential of half-maximal activation, Vmis the membrane potential, and k is the slope factor. For the analysis of the steady state inactivation, maximal current amplitudes during the tail were corrected for the closing during the hyperpolarizing step at very negative potentials as described previously.27 The corrected curves were fitted by a Boltzmann function. The inhibition of currents by droperidol was quantified by the reduction of the maximal current during the test pulse. The fractional block f was calculated by the following formula: f = 1 − (Imax, drug/Imax, control). Concentration-response curves were fitted by a Hill equation: f = 1/[1 + (IC50/c)h], where c is the concentration of droperidol and h is the Hill coefficient. The impact of droperidol on the APD at 90% repolarization (APD90) was quantified by the following formula: y = (APD90, drug/((APD90, control+ APD90, washout)/2), where APD90, controlis the APD90before application, APD90, drugis the APD90in the presence of droperidol, and APD90, washoutis the APD90after maximal possible washout of the drug.
Statistical Analysis
All data are given as mean ± SD; n values indicate the number of experiments. When only two groups were compared, statistical significance was calculated by the Student t  test, otherwise by one-way analysis of variance (ANOVA) followed by Bonferroni post hoc  test using Excel (Microsoft, Redmond, WA) or PRISM (GraphPad Software Inc., San Diego, CA). Where appropriate, paired t  tests were performed. Statistical significance was defined as P  < 0.05.
Computer Simulations of Cardiac Action Potentials
Computer simulations were conducted using a modified version of the Luo-Rudy dynamic model as previously described.28–30 The influence of droperidol on the AP was simulated assuming a reduction of IKrby 50% and by variable degrees of ICaLinhibition. Simulations were performed with a basic cycle length of 1,000 ms for 500 cycles.
Results
Effects of Droperidol on HERG and KCNQ1/KCNE1 Currents
The effect of droperidol on HERG and KCNQ1/KCNE1 currents was studied in oocytes injected with corresponding cRNA. Throughout all experiments, the holding potential was −80 mV, and repetitive pulses were applied to ensure steady state conditions.
Different pulse protocols were employed to assess the effect of droperidol on HERG currents: The ramp protocol was used to elicit a HERG current similar to that during a normal AP.20,31 Oocytes were depolarized to +60 mV and repolarized to −80 mV within 1 s, and the maximal current Imaxwas determined. Using square pulses, the cells were depolarized to +0 mV for 2 s, and tail currents were then recorded at −40 mV (Itail −40) or at −120 mV (Itail −120). Figure 1Ashows representative current traces elicited by the different pulse protocols under control conditions and after application of 1 μm droperidol. A strong suppression of HERG currents could be detected with each pulse protocol. The concentration dependent fractional inhibition of Imax, Itail −40, and Itail −120were described by Hill equations (fig. 1Band table 1). The IC50was in the range of 0.6–0.9 μm. The inhibition at a concentration of 1 μm droperidol did not significantly differ between the applied pulse protocols (statistically not significant; ANOVA). Therefore, this concentration was chosen to further analyze the effect of droperidol on channel gating properties. The Hill coefficients of all Hill equations were comparable.
Fig. 1. Effect of droperidol on human  ether-a-go-go  -related gene (HERG) and KCNQ1/KCNE1 currents. (  A  ) Representative HERG current traces and effect of 1 μm droperidol in oocytes (  upper panels  ) evoked by the pulse protocols shown in the  lower panels  . (  B  ) Concentration dependence of HERG channel block by droperidol. Inhibition was calculated as the reduction of the respective currents assessed by the three test protocols. Curves were fitted by Hill equations (parameters are shown in  table 1). Each data point represents 5–10 experiments. (  C  ) Representative KCNQ1/KCNE1 current traces evoked by the pulse protocol shown in the inset and the effect of 10 μm droperidol. 
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Fig. 1. Effect of droperidol on human  ether-a-go-go  -related gene (HERG) and KCNQ1/KCNE1 currents. (  A  ) Representative HERG current traces and effect of 1 μm droperidol in oocytes (  upper panels  ) evoked by the pulse protocols shown in the  lower panels  . (  B  ) Concentration dependence of HERG channel block by droperidol. Inhibition was calculated as the reduction of the respective currents assessed by the three test protocols. Curves were fitted by Hill equations (parameters are shown in  table 1). Each data point represents 5–10 experiments. (  C  ) Representative KCNQ1/KCNE1 current traces evoked by the pulse protocol shown in the inset and the effect of 10 μm droperidol. 
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Table 1. Inhibition of HERG Currents by Droperidol 
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Table 1. Inhibition of HERG Currents by Droperidol 
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To investigate the effect of droperidol on KCNQ1/KCNE1 currents, oocytes were depolarized to +60 mV for a duration of 5 s (Itest). Tail currents were recorded at −60 mV (Itail −60). Figure 1Cshows representative current traces under control conditions and after application of 10 μm droperidol. Whereas 1 μm droperidol did not influence the current amplitude of Itest(n = 4), 10 μm droperidol reduced Itestin two of five experiments by 3% and 5%. Itail −60was not affected by either concentration.
Effects of Droperidol on HERG Channel Gating
HERG Activation.
To analyze the influence of droperidol on the activation of HERG currents, the pulse protocol shown in figure 2Awas used. Depolarizing steps between −70 mV and +80 mV activated time-dependent outward currents. The amplitude of the outward currents at the end of the depolarizing pulse (Itest) increased with more positive voltage steps and reached a maximum value at approximately 0 mV. Depolarization to more positive values caused a current decrease. To visualize the current inhibition by droperidol, Itestwas normalized to the maximum control current and plotted against the activation potential under control conditions and in the presence of 1 μm droperidol (fig. 2B). Whereas droperidol strongly inhibited the amplitude of Itest, no influence on the voltage-dependence of activation was detected.
Fig. 2. Activation of human  ether-a-go-go  -related gene (HERG) currents and the influence of 1 μm droperidol. (  A  ) Representative current traces under control conditions and in the presence of 1 μm droperidol evoked by the depicted pulse protocol. (  B  ) Voltage dependence of Itestactivation and the influence of 1 μm droperidol. Itestwas normalized to the maximal current amplitude under control conditions. (  C  ) To visualize the effect of droperidol on the voltage-dependent activation of tail current amplitude, Itailwas normalized to the maximal tail current and fitted by Boltzmann functions (parameters are shown in  table 2). Droperidol significantly shifted the voltage of half-maximal activation V0.5toward more negative voltages and decreased the slope factor (  P  < 0.001). (  D  ) Inhibition of the tail current significantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, eight cells were investigated. 
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Fig. 2. Activation of human  ether-a-go-go  -related gene (HERG) currents and the influence of 1 μm droperidol. (  A  ) Representative current traces under control conditions and in the presence of 1 μm droperidol evoked by the depicted pulse protocol. (  B  ) Voltage dependence of Itestactivation and the influence of 1 μm droperidol. Itestwas normalized to the maximal current amplitude under control conditions. (  C  ) To visualize the effect of droperidol on the voltage-dependent activation of tail current amplitude, Itailwas normalized to the maximal tail current and fitted by Boltzmann functions (parameters are shown in  table 2). Droperidol significantly shifted the voltage of half-maximal activation V0.5toward more negative voltages and decreased the slope factor (  P  < 0.001). (  D  ) Inhibition of the tail current significantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, eight cells were investigated. 
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Table 2. Influence of Droperidol on HERG Channel Kinetic Parameters 
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Table 2. Influence of Droperidol on HERG Channel Kinetic Parameters 
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After the depolarizing step, the tail current Itailwas elicited by a repolarization to −40 mV. The amplitude of Itailincreased with depolarizing steps from −70 to +20 mV and reached a current plateau upon further depolarization (fig. 2C). Itailwas normalized to the maximum Itailof each cell and plotted against the depolarizing membrane potential. The resulting current-voltage curves (fig. 2C) were described by Boltzmann equations (see table 2for details). Droperidol significantly shifted the membrane potential of half-maximal activation (V0.5) toward negative potentials (P  < 0.001) and decreased the slope factor (P  < 0.001). The inhibition of Itailby droperidol significantly increased with depolarization of the membrane potential (P  < 0.001, ANOVA, n = 8; fig. 2D).
HERG Inactivation.
The influence of droperidol on the steady state inactivation was assessed by a three-step protocol (fig. 3A): The membrane was depolarized for 2 s to +40 mV, then pulses from −120 to +60 mV in steps of 10 mV were applied for 50 ms, and finally the membrane potential was kept at +40 mV for 1 s. Figure 3Ashows representative current traces under control conditions and after application of 1 μm droperidol. The magnitude of the resulting tail current at +40 mV was normalized to the maximum tail current and was plotted against the depolarizing voltage steps (fig. 3B). The parameters of the corresponding Boltzmann functions are given in table 2. Whereas droperidol caused a significant left shift of the inactivation midpoint (P  < 0.001), the slope factor was not influenced. The fractional inhibition of Itailsignificantly depended on the membrane potential (P  < 0.001, ANOVA, n = 7; fig. 3C). The inactivation time constants were analyzed using a three-step pulse protocol (data not shown): depolarization to 0 mV (2 s) followed by a repolarization to −80 mV for 50 ms and recording of the tail currents between −80 and +40 mV. The time constants of the monoexponential fits of the current decay under control conditions were similar to the ones found after application of 1 μm droperidol (droperidol, 18.0 ± 5.7 ms; control, 20.1 ± 6.2 ms at a pipette potential of 0 mV; n = 7).
Fig. 3. Inactivation of human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol. (  A  ) Representative current traces of HERG currents showing the steady state inactivation evoked by the depicted pulse protocol. (  B  ) Voltage dependence of steady state inactivation. The tail currents were normalized to the maximum tail currents and fitted by Boltzmann functions. The parameters of the Boltzmann fits are shown in  table 2. Droperidol caused a significant shift of the inactivation midpoint (  P  < 0.001) (  C  ) The inhibition of Itailsignificantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, 10 cells were investigated. 
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Fig. 3. Inactivation of human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol. (  A  ) Representative current traces of HERG currents showing the steady state inactivation evoked by the depicted pulse protocol. (  B  ) Voltage dependence of steady state inactivation. The tail currents were normalized to the maximum tail currents and fitted by Boltzmann functions. The parameters of the Boltzmann fits are shown in  table 2. Droperidol caused a significant shift of the inactivation midpoint (  P  < 0.001) (  C  ) The inhibition of Itailsignificantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, 10 cells were investigated. 
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HERG Deactivation.
The influence of droperidol on HERG deactivation was investigated using the following pulse protocol: depolarization to +60 mV (2 s) followed by repolarization steps to −120 to −50 mV (2 s). The current decay was best described by exponential functions with one or two time constants, respectively. There was no significant difference between the time constants under control conditions and in the presence of 1 μm droperidol (n = 8 oocytes investigated; ANOVA: statistically not significant; data not shown).
Time Dependence of Droperidol Inhibition.
The time dependence of HERG block was investigated with an “envelope of tails” protocol (fig. 4). The pulse protocol as well as representative current traces are depicted in figure 4A. After a depolarization to +40 mV for a duration (Δt) of 100–700 ms, tail currents were recorded at −40 mV. Fractional inhibition of the tail currents was calculated for each depolarization (fig. 4B). The time-dependent increase of inhibition followed a monoexponential function with a time constant of 186 ± 36 ms.
Fig. 4. Time dependence of droperidol block. (  A  ) Representative current recordings under control conditions and in the presence of 1 μm droperidol evoked by the “envelope of tails” protocol shown in the inset. (  B  ) Average inhibition of the tail current calculated for each depolarization period (Δt) in seven cells. (  C  ) The frequency dependence of human  ether-a-go-go  -related gene (HERG) inhibition by 1 μm droperidol was investigated after an incubation time of 10 min at −80 mV. Trains of 100 depolarizing pulses, as shown in the  inset  , were applied at a basic cycle length of 1 s (Δ; n = 5), 2 s (□; n = 4), 4 s (•; n = 4), or 10 s (○; n = 4). The resulting mean relative tail current amplitudes of the first 60 s are plotted  versus  time. The onset of inhibition significantly depended on the basic cycle length (  P  < 0.001, analysis of variance), whereas the steady state block was not dependent on the basic cycle length. 
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Fig. 4. Time dependence of droperidol block. (  A  ) Representative current recordings under control conditions and in the presence of 1 μm droperidol evoked by the “envelope of tails” protocol shown in the inset. (  B  ) Average inhibition of the tail current calculated for each depolarization period (Δt) in seven cells. (  C  ) The frequency dependence of human  ether-a-go-go  -related gene (HERG) inhibition by 1 μm droperidol was investigated after an incubation time of 10 min at −80 mV. Trains of 100 depolarizing pulses, as shown in the  inset  , were applied at a basic cycle length of 1 s (Δ; n = 5), 2 s (□; n = 4), 4 s (•; n = 4), or 10 s (○; n = 4). The resulting mean relative tail current amplitudes of the first 60 s are plotted  versus  time. The onset of inhibition significantly depended on the basic cycle length (  P  < 0.001, analysis of variance), whereas the steady state block was not dependent on the basic cycle length. 
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Frequency Dependence of Droperidol Inhibition.
The frequency dependence of HERG current inhibition by droperidol was investigated after the drug was allowed to wash in for 10 min at −80 mV (fig. 4C). HERG currents were activated by repetitive current pulses (depolarization to +20 mV of 300 ms duration, followed by a subsequent repolarization to −40 mV for 300 ms) at a basic cycle length of 1 s (n = 5), 2 s (n = 4), 4 s (n = 4), or 10 s (n = 4). After exposure to 1 μm droperidol, application of the pulse train at the different basic cycle lengths decreased tail current amplitude by 47 ± 5, 43 ± 10, 46 ± 12, or 44 ± 5% at a basic cycle length of 1, 2, 4, or 10 s, respectively. The time course of the inhibition was described by monoexponential functions with time constants of 1.2 ± 0.4, 1.9 ± 0.3, 4.3 ± 0.3, or 7.9 ± 1.3 s at a basic cycle length of 1, 2, 4, or 10 s, respectively. Although the development of block was faster at higher stimulation frequencies (P  < 0.001, ANOVA), the extent of steady state inhibition did not depend on the stimulation frequency.
Interaction Sites of Droperidol
The inhibitions of mutated HERG constructs (Y652A, F656A, T623A, S624A, and V625A) by droperidol were established to identify the molecular sites of interaction with HERG channels. The mutants S624A and Y652A show similar gating behavior as wild-type HERG channels. Inhibition by 1 μm droperidol was assessed using the three pulse protocols shown in figure 1A. The mutants T623A, V625A, and F656A gave rise to only small currents in response to either the ramp protocol or the protocol eliciting tail currents at −40 mV. Therefore, these mutants were investigated by assessing the inhibition of tail currents at −120 mV. Representative current recordings of wild-type HERG and mutant channels under control conditions and in the presence of 1 μm droperidol are shown in figure 5A. The residual tail currents at −120 mV are depicted in figure 5B. Inhibition of all mutants by 1 μm droperidol was significantly smaller than inhibition of wild-type channels (see also table 3).
Fig. 5. Molecular determinants of droperidol block. (  A  ) Representative traces of wild-type (wt) and mutant human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol evoked by the pulse protocol shown in the  inset  . (  B  ) Residual tail currents at −120 mV of the different HERG constructs in the presence of 1 μm droperidol. Parameters are given in  table 3. For each HERG mutant channel, 5–10 oocytes were investigated. 
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Fig. 5. Molecular determinants of droperidol block. (  A  ) Representative traces of wild-type (wt) and mutant human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol evoked by the pulse protocol shown in the  inset  . (  B  ) Residual tail currents at −120 mV of the different HERG constructs in the presence of 1 μm droperidol. Parameters are given in  table 3. For each HERG mutant channel, 5–10 oocytes were investigated. 
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Table 3. Inhibition of HERG Mutant Channels by 1 μm Droperidol 
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Table 3. Inhibition of HERG Mutant Channels by 1 μm Droperidol 
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Influence of Droperidol on APD of Guinea Pig Cardiac Myocytes
Figures 6A and Bshow representative APs recorded from isolated guinea pig left ventricular myocytes under control condition and in the presence of droperidol after an incubation time of 3 min. APs were elicited at 0.2 Hz by short depolarizing currents under steady state conditions. Figure 6Cvisualizes the influence of droperidol on APD90, and table 4summarizes AP details. Droperidol influenced neither the resting membrane potential nor the AP overshoot. At low concentrations (5–100 nm), droperidol increased APD in a concentration-dependent manner with a maximal prolongation of 17 ± 5% observed at a concentration of 100 nm. At a concentration of 300 nm, the AP prolongation was abolished, whereas even higher concentrations of up to 10 μm shortened APD compared with control.
Fig. 6. Concentration-dependent effects of droperidol on action potentials of guinea pig cardiac myocytes. (  A  ) Representative action potentials recorded under control conditions and after application of 10–100 nm droperidol. In this concentration range, droperidol increased the duration of the action potential in a concentration-dependent manner. (  B  ) Representative action potentials under control conditions and in the presence of 1 and 10 μm droperidol. Both concentrations shortened the action potential. (  C  ) Concentration-dependent effect of droperidol on the action potential duration at 90% repolarization. For each concentration, four to nine myocytes were investigated. Parameters are given in  table 4. *  P  < 0.05, **  P  < 0.01, ***  P  < 0.001, droperidol  versus  control, paired  t  test. APD90= action potential duration at 90% repolarization; Vm= resting membrane potential. 
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Fig. 6. Concentration-dependent effects of droperidol on action potentials of guinea pig cardiac myocytes. (  A  ) Representative action potentials recorded under control conditions and after application of 10–100 nm droperidol. In this concentration range, droperidol increased the duration of the action potential in a concentration-dependent manner. (  B  ) Representative action potentials under control conditions and in the presence of 1 and 10 μm droperidol. Both concentrations shortened the action potential. (  C  ) Concentration-dependent effect of droperidol on the action potential duration at 90% repolarization. For each concentration, four to nine myocytes were investigated. Parameters are given in  table 4. *  P  < 0.05, **  P  < 0.01, ***  P  < 0.001, droperidol  versus  control, paired  t  test. APD90= action potential duration at 90% repolarization; Vm= resting membrane potential. 
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Table 4. Influence of Droperidol on Action Potential Characteristics 
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Table 4. Influence of Droperidol on Action Potential Characteristics 
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Discussion
In the current study, the molecular mechanisms underlying inhibition of HERG channels by droperidol were characterized. The role in drug channel interaction of several amino acid residues located in the S6 domain and within the channel pore were established. Furthermore, possible consequences of this interaction for the excitability of isolated ventricular cardiomyocytes were elucidated.
Droperidol blocked HERG currents in Xenopus  oocytes with an IC50value between 0.6 and 0.9 μm. This value is well within the range of that reported for other high-affinity blockers such as dofetilide and E-4031 (0.4 and 0.6 μm, respectively).19 The difference between the value reported in the current study and the value reported by Drolet et al.  12 (approximately 0.03 μm in HEK293 cells) is most likely caused by differences in the expression systems.30 Droperidol did not significantly influence KCNQ1/KCNE1 currents, which confirms that droperidol specifically interacts with IKrbut not with IKs. Whereas droperidol potently inhibited the amplitude of HERG currents, it had only small effects on channel gating. Channel activation and steady state inactivation were slightly shifted toward negative potentials, whereas deactivation was not influenced. Current inhibition by droperidol significantly increased with the membrane potential and with increasing duration of current activation. Furthermore, the development of block depended on the stimulation frequency. These results indicate that the inhibition by droperidol may be state dependent, with a preference for open channels. Therefore, droperidol acts similarly to other high-affinity blockers of HERG channels.19,21 
Several residues located in the S6 domain (Tyr652, Phe656) and within the channel pore (Thr623, Ser624, Val625) are important for the interaction of high-affinity blockers with HERG channels.16,17,19,32 In the current study, these amino acid residues were identified to also mediate inhibition of HERG channels by droperidol. However, these mutations are known to alter channel gating behavior.16,20 Whereas S624A slows and V625A abolishes channel inactivation, the other mutants accelerate inactivation. Moreover, all mutations shift the voltage dependence of activation and/or inactivation. Although it cannot be excluded that the reduced inhibition by droperidol is related to changes in channel gating, this seems less likely. The reduction in inhibition is similar for all mutations despite the changes in gating being qualitatively different. The role of inactivation in HERG channel block is controversial: Abolishing C-type inactivation by introducing the double mutant HERGG628C/S631Creduces inhibition of HERG channel by blockers such as dofetilide33 and E-4031.34 However, other mutations affecting HERG inactivation yield inconsistent results regarding the role of inactivation for the inhibition by high-affinity blockers.35,36 Our results demonstrate that inhibition of HERG channels by droperidol is reduced to a similar extend by a mutation that accelerates inactivation (T623A) and by a mutation that abolishes inactivation (V625A). Reduced droperidol affinity of noninactivating HERG mutant channels may thus result from inactivation gating-associated reorientation of residues in the S6 domain that comprise the droperidol binding site rather than resulting from a direct effect of the inactivation. Taken together, our findings may, therefore, suggest that inactivation is neither a sufficient nor a necessary prerequisite for high-affinity block of HERG channels by droperidol.
Consistent with its potency to inhibit IKr, droperidol prolonged the APD in guinea pig cardiomyocytes. At concentrations between 5 and 100 nm, droperidol increased APD in a concentration-dependent manner with a maximal increase of approximately 20% at 100 nm. Further increases of the droperidol concentration of 300 nm or greater, however, attenuated the AP prolongation and reversed the effect at even higher concentrations. This dual concentration-dependent effect on APD of ventricular cardiomyocytes may explain why some authors found AP shortening,8,9 whereas others found AP prolongation11,12 or no effect on APD.10 The potency of droperidol to prolong APD and to induce EADs is a matter of debate.8–12 Droperidol at antiemetic concentrations is estimated to reach free plasma concentrations of approximately 30 nm.37–39 At this concentration, IKrwould be inhibited by a maximum of 50%.12 Assuming that droperidol at these plasma concentrations exclusively inhibits HERG currents, computational modeling based on the Luo-Rudy dynamic model would predict APD prolongation and induction of EADs (fig. 7A). However, EADs were not observed in our experiments with ventricular cardiomyocytes. Furthermore, EADs and severe ventricular arrhythmias have not been reported during the antiemetic application of droperidol.2,4 Our results may, therefore, suggest that despite being a high-affinity blocker of HERG channels, additional pharmacologic effects of droperidol may counterbalance these proarrhythmic effects.
Fig. 7. Computational modeling of cardiac action potentials of epicardial, midmyocardial, and endocardial myocytes using the Luo-Rudy dynamic model. (  A  ) Simulation of ventricular action potentials under standard conditions (control) and with 50% inhibition of IKr. Action potential prolongation was larger in endocardial (20 ms or approximately 9%) than in epicardial myocytes (13 ms or approximately 7%). In midmyocardial cells, repolarization did not occur within one stimulation interval, and early afterdepolarizations developed. (  B  ) Simulation of control action potentials and with 50% inhibition of IKrand 40% inhibition of ICaL. While the action potential duration in midmyocardial cells was prolonged by 28 ms (approximately 10%), such a combined channel block abolished the prolongation of the action potential duration in endocardial and epicardial cells. No early afterdepolarizations developed. All simulations were performed with a basic cycle length of 1,000 ms for 500 cycles at body temperature. 
Image Not Available
Fig. 7. Computational modeling of cardiac action potentials of epicardial, midmyocardial, and endocardial myocytes using the Luo-Rudy dynamic model. (  A  ) Simulation of ventricular action potentials under standard conditions (control) and with 50% inhibition of IKr. Action potential prolongation was larger in endocardial (20 ms or approximately 9%) than in epicardial myocytes (13 ms or approximately 7%). In midmyocardial cells, repolarization did not occur within one stimulation interval, and early afterdepolarizations developed. (  B  ) Simulation of control action potentials and with 50% inhibition of IKrand 40% inhibition of ICaL. While the action potential duration in midmyocardial cells was prolonged by 28 ms (approximately 10%), such a combined channel block abolished the prolongation of the action potential duration in endocardial and epicardial cells. No early afterdepolarizations developed. All simulations were performed with a basic cycle length of 1,000 ms for 500 cycles at body temperature. 
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It is well known that several HERG blockers exhibit additional effects on Ca2+channels.40–43 Because ICaLis involved in the development of EADs,44–48 blockade of this current has repeatedly been suggested to diminish the proarrhythmic effects induced by inhibition of HERG channels.42,49,50 The calcium channel antagonist verapamil also is a highly potent inhibitor of IKrand specifically interacts with HERG channels.40 Despite its specific effects on HERG channels, verapamil abolishes EADs and does not induce torsades de pointes ventricular arrhythmia.41–43,45,51–54 Although direct effects of droperidol on ICaLare unknown, indirect evidence suggests that droperidol interacts with ICaL.8,9,11,55 Droperidol has recently been reported to decrease the potassium chloride-induced increase of Ca2+concentration in myocytes, indicative of an interaction with ICaL.55 Inhibition of ICaLwould explain the AP shortening by droperidol reported in the current study and previous studies.8,9 Therefore, we simulated 50% IKrblock with different degrees of ICaLblock. Computational modeling indicates that inhibition of ICaLby 40% would prevent the occurrence of EADs (fig. 7B). It may, therefore, be hypothesized that concomitant inhibition of IKrand ICaLprevents induction of EADs by droperidol in cardiomyocytes. Such a dual mechanism has been established for verapamil and also for the neuroleptic agent risperidone.43,50 Furthermore, this dual effect may offer an explanation why severe ventricular cardiac arrhythmia have not been reported after application of droperidol at antiemetic doses.2,4 
In conclusion, this study provides evidence that droperidol is a high-affinity blocker of HERG channels causing only minor alterations of channel gating. The residues Thr623, Ser624, Val625, Tyr652, and Phe656 are important for the drug effect. Despite this molecular mode of interaction, droperidol does not induce EADs in ventricular cardiomyocytes. Computational modeling allows us to hypothesize that interaction with other depolarizing currents such as ICaLmay explain why droperidol at antiemetic concentrations prolongs the APD without inducing EADs. This hypothesis warrants further investigation.
The authors thank Olaf Pongs, Ph.D. (Director of the Institute of Neural Signal Transduction, University Medical Center, Hamburg-Eppendorf, Germany), for his continuous support.
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Fig. 1. Effect of droperidol on human  ether-a-go-go  -related gene (HERG) and KCNQ1/KCNE1 currents. (  A  ) Representative HERG current traces and effect of 1 μm droperidol in oocytes (  upper panels  ) evoked by the pulse protocols shown in the  lower panels  . (  B  ) Concentration dependence of HERG channel block by droperidol. Inhibition was calculated as the reduction of the respective currents assessed by the three test protocols. Curves were fitted by Hill equations (parameters are shown in  table 1). Each data point represents 5–10 experiments. (  C  ) Representative KCNQ1/KCNE1 current traces evoked by the pulse protocol shown in the inset and the effect of 10 μm droperidol. 
Image Not Available
Fig. 1. Effect of droperidol on human  ether-a-go-go  -related gene (HERG) and KCNQ1/KCNE1 currents. (  A  ) Representative HERG current traces and effect of 1 μm droperidol in oocytes (  upper panels  ) evoked by the pulse protocols shown in the  lower panels  . (  B  ) Concentration dependence of HERG channel block by droperidol. Inhibition was calculated as the reduction of the respective currents assessed by the three test protocols. Curves were fitted by Hill equations (parameters are shown in  table 1). Each data point represents 5–10 experiments. (  C  ) Representative KCNQ1/KCNE1 current traces evoked by the pulse protocol shown in the inset and the effect of 10 μm droperidol. 
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Fig. 2. Activation of human  ether-a-go-go  -related gene (HERG) currents and the influence of 1 μm droperidol. (  A  ) Representative current traces under control conditions and in the presence of 1 μm droperidol evoked by the depicted pulse protocol. (  B  ) Voltage dependence of Itestactivation and the influence of 1 μm droperidol. Itestwas normalized to the maximal current amplitude under control conditions. (  C  ) To visualize the effect of droperidol on the voltage-dependent activation of tail current amplitude, Itailwas normalized to the maximal tail current and fitted by Boltzmann functions (parameters are shown in  table 2). Droperidol significantly shifted the voltage of half-maximal activation V0.5toward more negative voltages and decreased the slope factor (  P  < 0.001). (  D  ) Inhibition of the tail current significantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, eight cells were investigated. 
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Fig. 2. Activation of human  ether-a-go-go  -related gene (HERG) currents and the influence of 1 μm droperidol. (  A  ) Representative current traces under control conditions and in the presence of 1 μm droperidol evoked by the depicted pulse protocol. (  B  ) Voltage dependence of Itestactivation and the influence of 1 μm droperidol. Itestwas normalized to the maximal current amplitude under control conditions. (  C  ) To visualize the effect of droperidol on the voltage-dependent activation of tail current amplitude, Itailwas normalized to the maximal tail current and fitted by Boltzmann functions (parameters are shown in  table 2). Droperidol significantly shifted the voltage of half-maximal activation V0.5toward more negative voltages and decreased the slope factor (  P  < 0.001). (  D  ) Inhibition of the tail current significantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, eight cells were investigated. 
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Fig. 3. Inactivation of human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol. (  A  ) Representative current traces of HERG currents showing the steady state inactivation evoked by the depicted pulse protocol. (  B  ) Voltage dependence of steady state inactivation. The tail currents were normalized to the maximum tail currents and fitted by Boltzmann functions. The parameters of the Boltzmann fits are shown in  table 2. Droperidol caused a significant shift of the inactivation midpoint (  P  < 0.001) (  C  ) The inhibition of Itailsignificantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, 10 cells were investigated. 
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Fig. 3. Inactivation of human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol. (  A  ) Representative current traces of HERG currents showing the steady state inactivation evoked by the depicted pulse protocol. (  B  ) Voltage dependence of steady state inactivation. The tail currents were normalized to the maximum tail currents and fitted by Boltzmann functions. The parameters of the Boltzmann fits are shown in  table 2. Droperidol caused a significant shift of the inactivation midpoint (  P  < 0.001) (  C  ) The inhibition of Itailsignificantly depended on the membrane potential (  P  < 0.001, analysis of variance). For each parameter, 10 cells were investigated. 
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Fig. 4. Time dependence of droperidol block. (  A  ) Representative current recordings under control conditions and in the presence of 1 μm droperidol evoked by the “envelope of tails” protocol shown in the inset. (  B  ) Average inhibition of the tail current calculated for each depolarization period (Δt) in seven cells. (  C  ) The frequency dependence of human  ether-a-go-go  -related gene (HERG) inhibition by 1 μm droperidol was investigated after an incubation time of 10 min at −80 mV. Trains of 100 depolarizing pulses, as shown in the  inset  , were applied at a basic cycle length of 1 s (Δ; n = 5), 2 s (□; n = 4), 4 s (•; n = 4), or 10 s (○; n = 4). The resulting mean relative tail current amplitudes of the first 60 s are plotted  versus  time. The onset of inhibition significantly depended on the basic cycle length (  P  < 0.001, analysis of variance), whereas the steady state block was not dependent on the basic cycle length. 
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Fig. 4. Time dependence of droperidol block. (  A  ) Representative current recordings under control conditions and in the presence of 1 μm droperidol evoked by the “envelope of tails” protocol shown in the inset. (  B  ) Average inhibition of the tail current calculated for each depolarization period (Δt) in seven cells. (  C  ) The frequency dependence of human  ether-a-go-go  -related gene (HERG) inhibition by 1 μm droperidol was investigated after an incubation time of 10 min at −80 mV. Trains of 100 depolarizing pulses, as shown in the  inset  , were applied at a basic cycle length of 1 s (Δ; n = 5), 2 s (□; n = 4), 4 s (•; n = 4), or 10 s (○; n = 4). The resulting mean relative tail current amplitudes of the first 60 s are plotted  versus  time. The onset of inhibition significantly depended on the basic cycle length (  P  < 0.001, analysis of variance), whereas the steady state block was not dependent on the basic cycle length. 
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Fig. 5. Molecular determinants of droperidol block. (  A  ) Representative traces of wild-type (wt) and mutant human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol evoked by the pulse protocol shown in the  inset  . (  B  ) Residual tail currents at −120 mV of the different HERG constructs in the presence of 1 μm droperidol. Parameters are given in  table 3. For each HERG mutant channel, 5–10 oocytes were investigated. 
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Fig. 5. Molecular determinants of droperidol block. (  A  ) Representative traces of wild-type (wt) and mutant human  ether-a-go-go  -related gene (HERG) currents under control conditions and in the presence of 1 μm droperidol evoked by the pulse protocol shown in the  inset  . (  B  ) Residual tail currents at −120 mV of the different HERG constructs in the presence of 1 μm droperidol. Parameters are given in  table 3. For each HERG mutant channel, 5–10 oocytes were investigated. 
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Fig. 6. Concentration-dependent effects of droperidol on action potentials of guinea pig cardiac myocytes. (  A  ) Representative action potentials recorded under control conditions and after application of 10–100 nm droperidol. In this concentration range, droperidol increased the duration of the action potential in a concentration-dependent manner. (  B  ) Representative action potentials under control conditions and in the presence of 1 and 10 μm droperidol. Both concentrations shortened the action potential. (  C  ) Concentration-dependent effect of droperidol on the action potential duration at 90% repolarization. For each concentration, four to nine myocytes were investigated. Parameters are given in  table 4. *  P  < 0.05, **  P  < 0.01, ***  P  < 0.001, droperidol  versus  control, paired  t  test. APD90= action potential duration at 90% repolarization; Vm= resting membrane potential. 
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Fig. 6. Concentration-dependent effects of droperidol on action potentials of guinea pig cardiac myocytes. (  A  ) Representative action potentials recorded under control conditions and after application of 10–100 nm droperidol. In this concentration range, droperidol increased the duration of the action potential in a concentration-dependent manner. (  B  ) Representative action potentials under control conditions and in the presence of 1 and 10 μm droperidol. Both concentrations shortened the action potential. (  C  ) Concentration-dependent effect of droperidol on the action potential duration at 90% repolarization. For each concentration, four to nine myocytes were investigated. Parameters are given in  table 4. *  P  < 0.05, **  P  < 0.01, ***  P  < 0.001, droperidol  versus  control, paired  t  test. APD90= action potential duration at 90% repolarization; Vm= resting membrane potential. 
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Fig. 7. Computational modeling of cardiac action potentials of epicardial, midmyocardial, and endocardial myocytes using the Luo-Rudy dynamic model. (  A  ) Simulation of ventricular action potentials under standard conditions (control) and with 50% inhibition of IKr. Action potential prolongation was larger in endocardial (20 ms or approximately 9%) than in epicardial myocytes (13 ms or approximately 7%). In midmyocardial cells, repolarization did not occur within one stimulation interval, and early afterdepolarizations developed. (  B  ) Simulation of control action potentials and with 50% inhibition of IKrand 40% inhibition of ICaL. While the action potential duration in midmyocardial cells was prolonged by 28 ms (approximately 10%), such a combined channel block abolished the prolongation of the action potential duration in endocardial and epicardial cells. No early afterdepolarizations developed. All simulations were performed with a basic cycle length of 1,000 ms for 500 cycles at body temperature. 
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Fig. 7. Computational modeling of cardiac action potentials of epicardial, midmyocardial, and endocardial myocytes using the Luo-Rudy dynamic model. (  A  ) Simulation of ventricular action potentials under standard conditions (control) and with 50% inhibition of IKr. Action potential prolongation was larger in endocardial (20 ms or approximately 9%) than in epicardial myocytes (13 ms or approximately 7%). In midmyocardial cells, repolarization did not occur within one stimulation interval, and early afterdepolarizations developed. (  B  ) Simulation of control action potentials and with 50% inhibition of IKrand 40% inhibition of ICaL. While the action potential duration in midmyocardial cells was prolonged by 28 ms (approximately 10%), such a combined channel block abolished the prolongation of the action potential duration in endocardial and epicardial cells. No early afterdepolarizations developed. All simulations were performed with a basic cycle length of 1,000 ms for 500 cycles at body temperature. 
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Table 1. Inhibition of HERG Currents by Droperidol 
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Table 1. Inhibition of HERG Currents by Droperidol 
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Table 2. Influence of Droperidol on HERG Channel Kinetic Parameters 
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Table 2. Influence of Droperidol on HERG Channel Kinetic Parameters 
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Table 3. Inhibition of HERG Mutant Channels by 1 μm Droperidol 
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Table 3. Inhibition of HERG Mutant Channels by 1 μm Droperidol 
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Table 4. Influence of Droperidol on Action Potential Characteristics 
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Table 4. Influence of Droperidol on Action Potential Characteristics 
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