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(Circulation. 1996;94:817-823.)
© 1996 American Heart Association, Inc.

Articles

HERG, a Primary Human Ventricular Target of the Nonsedating Antihistamine Terfenadine

Mary-Louise Roy, PhD; Robert Dumaine, PhD; Arthur M. Brown, MD, PhD

the Rammelkamp Center for Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio.

Correspondence to Arthur M. Brown MD, PhD, Rammelkamp Center for Research, MetroHealth Medical Center, Case Western Reserve University School of Medicine, 2500 MetroHealth Dr, Cleveland, OH 44109-1998. E-mail abrown@mhnet.mhmc.org.

	Abstract

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BackgroundAdministration of the antihistamine terfenadine (Seldane) to patients may result in acquired long QT syndrome and ventricular arrhythmias. One human cardiac target is Kv1.5, which expresses the ultrarapid outward K⁺ current (I_Kur) in atrium but may play only a minor role in ventricle. Another possible target is HERG, the human ether-a-go-go–related gene that expresses a delayed rectifier current (I_Kr) in human ventricle and produces hereditary long QT syndrome when defective.

Methods and ResultsWe therefore heterologously expressed Kv1.5 and HERG in Xenopus oocytes to compare the sensitivity of each to terfenadine. We found that HERG was 10 times more sensitive than Kv1.5 to terfenadine block. The apparent K_d values for HERG and Kv1.5 currents were 350 nmol/L and 2.7 µmol/L, respectively. These K_d values compare well with values reported for terfenadine block of I_Kr and I_Kur currents in human atrial myocytes. The K_d value for HERG block is relevant to the toxicity of the antihistamine, since the clinical terfenadine concentrations in human plasma may reach the 100 nmol/L range.

Conclusions Terfenadine carboxylate, the major metabolite of terfenadine, does not block either HERG or Kv1.5, which agrees with the hypothesis that the buildup of parent terfenadine is the likely explanation for its cardiotoxicity. We propose that the blocking of HERG by terfenadine explains the acquired long QT syndrome. HERG is likely to be the primary target for the known cardiotoxic effects of other, related antihistamines.

Key Words: terfenadine • ketoconazole • oocytes • delayed rectifier

	Introduction

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Antihistamines that block H1 receptors are prescribed for relief of symptoms from upper respiratory infections or allergy. The antihistamine side effect of excessive sedation prompted the search for newer antihistamines that did not cause this reaction. Terfenadine was the first of this class of drugs and in 1991 was the ninth–most frequently prescribed drug in the United States.¹ In the late 1980s, frequent reports of syncope, prolonged QT_c, and torsades de pointes began to appear.^{2 3 4 5 6} Symptoms were associated with overdose, hepatic disease, and inhibition of CYP3A4-P450 by conazoles and macrolide antibiotics.^{2 7} Other antihistamines, such as astemizole, have had similar effects.⁸ As a result, the US Food and Drug Administration now requires that physicians be advised directly as well as by warnings on drug packaging in bold, black type of the potentially adverse cardiac effects of these drugs.

At concentrations relevant to its toxicity in humans, terfenadine blocks the delayed rectifier (I_K) current^{9 10 11 12 13 14 15 16 17} in cat ventricle.¹⁸ At these concentrations, it also blocks human cardiac Kv1.5, the source of I_Kur in atrium^{17 18} but not in ventricle. At higher concentrations, terfenadine blocks I_Kur and I_Kr in human atrial myocytes.^{19 20}

Thus, neither the effects in cat ventricle nor those in human atrium truly localize the molecular target in human ventricle. We have accomplished such localization by comparing the effects of terfenadine on Kv1.5 and HERG, the human ether-a-go-go–related gene.^{21 22 23} HERG is highly relevant because it expresses I_Kr and is defective in hereditary long QT syndrome.^{22 24} We found that HERG was 10 times more sensitive to terfenadine blockade than Kv1.5 when both proteins were heterologously expressed in Xenopus oocytes. Therefore, HERG is the primary known target for the cardiotoxicity of terfenadine and, by extension, other related antihistamines.

	Methods

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Molecular Biology
The HERG clone was a gift from Drs M.T. Keating and M. Sanguinetti, and the full-length cDNA was cloned into the pSP64 transcription vector as previously described.²² Amplification of cDNA was obtained by transformation into E. coli and overnight incubation at 37°C. HERG cDNA was linearized by digestion with EcoR1 for runoff transcription with the SP6 mMessage mMachine in vitro transcription kit (Ambion). Kv1.5 was previously cloned in our laboratory¹⁷ and subcloned into A⁺-pCRII.²⁵ The final cRNA product was resuspended in 0.1 mol/L KCl and stored at -80°C. The cRNA was diluted to the desired concentration (5 to 150 pg/nL) immediately before oocyte injection. Stages V to VI Xenopus oocytes were defolliculated by collagenase treatment (2 mg/mL for 1.5 hours) in a Ca²⁺-free buffer solution containing (mmol/L) NaCl 82.5, KCl 2.5, MgCl₂ 1, HEPES 5, and gentamicin 100 µg/mL, pH 7.6 (NaOH-HCl). The defolliculated oocytes were injected with 46 nL cRNA solution (0.1 mol/L KCl) and incubated at 19°C in culture medium containing (mmol/L) NaCl 100, KCl 2, CaCl₂ 1.8, MgCl₂ 1, HEPES 5, pyruvic acid 2.5, and gentamicin 100 µg/mL, pH 7.6. Electrophysiological measurements were made 3 to 7 days after cRNA injection. All experiments were conducted in accordance with institutional animal care committee regulations.

Electrophysiology
Whole-cell currents were recorded from Xenopus oocytes by use of the conventional two-microelectrode voltage-clamp technique. Beveled microelectrodes were filled with a solution of 3 mol/L KCl, 10 mmol/L HEPES, and 10 mmol/L EGTA, pH 7.4 (Tris) to give a low tip resistance of 0.2 to 0.5 M. Oocytes were placed in a chamber and perfused with Ringer's solution containing (mmol/L) NaCl 120, KCl 2.5, CaCl₂ 1.1, EGTA 1.0, and HEPES-acid 10, pH 7.2 (NaOH). A stock solution of terfenadine (50 mmol/L, Sigma Chemical Co) was prepared in DMSO and diluted to the desired test concentrations with bath solution. To avoid artifacts, the portion of DMSO in the perfusing solution was never allowed to exceed 0.2% (v/v).

Current records were amplified with the use of a Warner oocyte clamp (OC-725A) and low-pass filtered at 3 kHz (-3 dB, 4-pole Bessel filter, Wavetech, model 432). Data were stored on the hard disk of a 486 IBM compatible computer for off-line analysis. All data acquisition and analysis were done with the suite of PCLAMP programs (Axon Instruments). Currents were recorded at room temperature, and experiments in which the holding current was >-0.2 µA at a holding level of -90 mV were excluded from analysis. Protocols are as described in the figure legends.

	Results

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Two-electrode voltage-clamp recordings of HERG currents revealed characteristic properties, as previously reported.^{22 23} Briefly, HERG current was activated at a potential >-40 mV by an 800-ms depolarizing pulse from a holding potential of -80 mV. The steady-state I/V had a typical bell-shaped waveform that peaked at 0 mV (Fig 1A and 1C), which closely matched the voltage dependence of the dofetilide-sensitive I_Kr found in human atrial¹⁹ and in guinea pig ventricular¹³ myocytes. Activation, measured as peak tail current after the stimulating pulse, reached a plateau for test potentials positive to 10 mV (Figs 1D and 2C).

View larger version (26K): [in this window] [in a new window]   Figure 1. HERG expression in Xenopus oocytes and terfenadine effects on whole-cell current. A, Representative family of current recordings generated from a holding potential of -80 mV and step pulses from -60 to +40 mV in 10-mV increments. Note the characteristic tail current and the rectification of outward current for pulses positive to 0 mV. B, Same oocyte after treatment with 2 µmol/L terfenadine, which reduces both steady-state and tail currents. The resulting I/Vs for steady-state (C) and tail (D) currents demonstrate the reduction of HERG current by terfenadine. Vm indicates the test potential. Open symbols indicate control; solid symbols, use of 2 µmol/L terfenadine. E, Dose-response curves for both steady-state and tail HERG currents, which generated Kd values of 350 and 391 nmol/L, respectively. F, Additional use-dependent block of HERG currents was not noted after repetitive pulses to +10 mV at 0.2 and 0.5 Hz from a holding potential of -80 mV. Open symbols indicate control; solid symbols, treatment with 10 µmol/L terfenadine; triangles, 0.2 Hz; inverted triangles, 0.5 Hz.

View larger version (25K): [in this window] [in a new window]   Figure 2. Terfenadine block of HERG steady-state and tail currents is not voltage-dependent. A, Relative I/Vs for steady-state HERG (current) are shown for control (n=5, ) and for treatment with 0.5 µmol/L (n=4, ) and 2.0 µmol/L terfenadine (n=6, ). B, Maximal steady-state current amplitudes after treatment with terfenadine (I peak terfenadine) are divided by the maximal control current (I peak) and plotted for each test potential. Data were fitted by a linear regression with comparable slopes of 2.5x10-4 and 2.6x10-4 mV-1 for 0.5 and 2.0 µmol/L, respectively. C, Tail-current amplitudes are similarly plotted. The linear regression line in D had slopes of -6.8x10 -4 and -7.8x10-4 mV-1 for 0.5 and 2.0 µmol/L terfenadine, respectively. Terfenadine block of HERG tail and steady-state currents is not significantly altered by voltage. Vm indicates the test potential.

Addition of 2 µmol/L terfenadine blocked the steady-state maximum current at 0 mV by 65% (Fig 1B and 1C). Terfenadine block was dose dependent and equally potent on the maximal steady-state and tail currents; its K_d values were 350 and 390 nmol/L, respectively, when measured by use of a 1:1 binding isotherm to fit the relative current amplitudes (Fig 1E). The solid lines in Fig 1E were generated by fitting data with a modified Hill equation and adjusting the asymptotic minimal values to 0.13 and 0.20 for steady-state and tail currents, respectively. In our recordings, HERG-rectifying kinetics and characteristic tail current were still observable at concentrations of 10 and 100 µmol/L, which suggests either that terfenadine is a partial blocker of HERG or that the oocyte lipid membrane, which is positively charged at physiological pH, is not easily permeable to terfenadine.

The block of HERG current reached a steady-state level within 7 minutes when the oocytes were superfused with Ringer's solution containing terfenadine (3 mL/min). To monitor the onset of block, we applied an 800-ms stimulus to 0 mV from a holding potential of -80 mV every 30 s. When a steady state was reached, we applied a pulsed stimulus at frequencies from 0.1 to 0.5 Hz to test for a use-dependent block. Although a small, residual portion of activated channels was carried from one pulse to the other at higher frequencies, as noted by a positive shift in the holding current, the relative decrease in active current amplitude remained the same with or without terfenadine (Fig 1F). We did not observe a use-dependent block in the range of frequencies studied.

We next considered the possibility of a voltage-dependent block by plotting the I/V curves for different concentrations of terfenadine. When we normalized the individual currents to the amplitude of their respective peak I/Vs, we did not find significant changes in the morphology of the I/V (Fig 2A and 2C). The fraction of peak current blocked by terfenadine was not statistically significant between -20 and +30 mV for both the steady-state and tail currents (Fig 2C and 2D), which indicated that the block was not voltage dependent. Changing the holding potential from -80 to -60 mV did not enhance the block.

We tested for a gating-dependent block by looking for changes in the waveform of HERG current at 0 and +40 mV in the presence of 2 µmol/L terfenadine (Fig 3A and 3B). When we normalized the currents to their respective maximal steady-state amplitudes (Fig 3C and 3D), we did not find significant waveform changes between the control and terfenadine-treated currents. We therefore concluded that terfenadine primarily produced a tonic block in the resting state.

View larger version (12K): [in this window] [in a new window]   Figure 3. Terfenadine does not alter HERG current kinetics. A, Voltage steps from -80 to 0 mV are shown under control and 2 µmol/L terfenadine–treated (*) conditions. B, Voltage step from -80 to +40 mV under similar conditions. C and D, Current traces are normalized and superimposed, with no significant change in kinetics.

Terfenadine has been shown to block Kv1.5 (a member of the Kv1 subfamily of voltage-dependent K⁺ channels) in human atrial myocytes and in the human embryonic kidney cell expression system; it has a K_d value in the micromolar range when applied extracellularly.^{19 26} We heterologously expressed Kv1.5 in Xenopus oocytes to compare the blockade by terfenadine in the same expression system. The current recorded when we applied a series of 350-ms depolarizing steps of 5 mV from a holding potential of -60 mV (Fig 4A) had a threshold of -20 mV and increased linearly at more depolarized potentials (Fig 4C), as previously described.^{17 19 26 27}

View larger version (25K): [in this window] [in a new window]   Figure 4. Terfenadine block of Kv1.5 current in Xenopus oocytes. A, Representative family of current recordings generated from a holding potential of -60 mV and step pulses from -40 to +50 mV in 5-mV increments. B, 10 µmol/L terfenadine reduces steady-state currents by 65%. C, I/V for A and B shows the current reductions by terfenadine. Vm indicates the test potential. D, Dose-response curve for the terfenadine block of Kv1.5 channels shows Kd values of 2.6 and 4.0 µmol/L for steady-state and peak currents, respectively (n=6). E, Additional use-dependent block of Kv1.5 currents was not noted after repetitive pulses to 0 mV at 0.2 and 0.5 Hz from a holding potential of -50 mV. Open symbols indicate control; solid symbols, use of 10 µmol/L terfenadine; triangles, 0.2 Hz, inverted triangles, 0.5 Hz.

Bath application of 10 µmol/L terfenadine produced a dose-dependent block with a 60% reduction of steady-state current (Fig 4C). The steady-state block was reached after 20 minutes. The dose-response curve for steady-state current was fitted by use of a Hill equation with a K_d value of 2.6 µmol/L (Fig 4D). As with HERG, we did not observe use-dependent block of terfenadine when the stimulus frequency was varied from 0.2 to 1 Hz (Fig 4E).

We also did not find significant morphological changes in the steady-state I/V between control and terfenadine-treated cells, apart from a monotonic reduction of current (Fig 5A). The fraction of peak current blocked by terfenadine was not statistically different for test potentials between 0 and 50 mV. Furthermore, changing the holding potential from -60 to -20 mV did not produce additional block. The block on Kv1.5 had two components: a tonic block, given by the reduction of the peak current, and a gating-dependent block that appeared after the channels were opened as faster inactivation (Fig 6). To assess the voltage dependence of the block, which developed after the channels were opened, we plotted the difference between the peak and the steady-state currents relative to the peak control current. As Fig 5C shows, extracellular application of terfenadine induced an extra, voltage-dependent block that was linked to the opening of the channels. A linear regression on the data points from 25 to 40 mV gave similar slopes. When higher (>10 µmol/L) concentrations of terfenadine were used, the voltage dependence of the extra block between 0 and 20 mV deviated from the regression line, which suggests a rate-limiting action of the activation kinetic on the onset of the extra block. These results show a 10-fold lower affinity and voltage-dependent block of Kv1.5 current by terfenadine relative to HERG.

View larger version (16K): [in this window] [in a new window]   Figure 5. Terfenadine block of Kv1.5 channels is not voltage dependent. A, Averaged relative steady-state (SS) current amplitudes for control (n=6, ) and for 2 (n=4, ) and 10 µmol/L (n=4, ) terfenadine. B, peak Kv1.5 currents with terfenadine (I peak terfenadine) are normalized to peak control current amplitude (I peak) for each test potential. Linear regression (solid lines) gave slopes of 1.4x10-4 mV-1 (at 2 µmol/L terfenadine) and 10x10-4 mV-1 (at 10 µmol/L terfenadine). C, Difference between peak and steady-state terfenadine currents (Delta) was normalized to the peak maximal control current (I max) and plotted against test potentials. Linear regressions (solid lines) returned slopes of 5.4x10-4 mV-1 (control), 2.0x10-3 mV-1 (2 µmol/L terfenadine), and 2.5x10-3 mV-1 (10 µmol/L terfenadine). Vm indicates the test potentials.

View larger version (9K): [in this window] [in a new window]   Figure 6. Terfenadine alters Kv1.5 current kinetics. A, Kv1.5 currents were recorded with the use of a 2-s test pulse to +40 mV from a holding potential of -60 mV under control conditions and in the presence of 100 µmol/L terfenadine. B, Current inactivation under control conditions was best fitted by single exponential decay, with a time constant () of 1124 ms. C, After terfenadine exposure, inactivation was faster and best fitted with a sum of two exponentials with time constants of 1124 (f) and 52 ms (s).

With 100 µmol/L terfenadine in the bath, the time course of the current changed from a monoexponential decay with a time constant of =1124 ms to a biexponential decay with fast and slow components of _f=52 ms (30%) and _s=1124 ms (70%), respectively. The appearance of a faster component, combined with the deviation of the extra block from the regression line for low test potentials and high concentrations of terfenadine, strongly suggest a block in the open state.

Since cardiotoxicity is thought to occur through the buildup of terfenadine after alteration of its hepatic metabolism or overdose, we tested the action of one of its major metabolites, terfenadine carboxylate (MDL16,455), on both channels. Bath application of 500 µmol/L MDL16,455 did not produce a significant block of either HERG (n=4) or Kv1.5 (n=5, Fig 7), a result consistent with cardiotoxicity such as results from increased plasmatic concentrations of terfenadine. The small reduction in tail current observed for HERG (Fig 7B) is probably due to a change in potassium reversal potential, since the cell was maintained in the perfusion chamber and stimulated for 30 minutes between measurements.

View larger version (25K): [in this window] [in a new window]   Figure 7. Terfenadine carboxylate did not block Kv1.5 or HERG. A, Kv1.5 currents were recorded by use of the same pulse protocol as in Fig 4A, under control conditions and after 25 minutes in the presence of 500 µmol/L MDL16,455. B, HERG currents were recorded by use of the protocol described in Fig 1A. Terfenadine carboxylate, at a concentration roughly 10 000-fold higher than the Kd value for terfenadine block on HERG, did not significantly reduce Kv1.5 or HERG currents.

Discussion
We found HERG to be 10-fold more sensitive than Kv1.5 to terfenadine block when both channels were expressed heterologously in Xenopus oocytes. The apparent K_d values for HERG (350 nmol/L) and Kv1.5 (2.7 µmol/L) compare well with the results from Crumb et al¹⁹ on I_Kr and I_Kur from human atrial myocytes when terfenadine was applied extracellularly.

The biophysical and pharmacological properties of HERG have been reported to fit closely those of I_Kr in ventricular cardiomyocytes.^{19 22 28} While an absence of E4031 sensitivity (a potent blocker of I_Kr) has been reported,²² further studies have shown that HERG is sensitive to extracellular application of dofetilide in the micromolar range when expressed heterologously in Xenopus oocytes.²³ We found HERG to be sensitive to dofetilide in the nanomolar range in excised, inside-out patches (J. Kiehn, MD, unpublished data, 1995), and a recent article²⁹ reported an IC₅₀ of 123±12 nmol/L for MK-499, another class III antiarrhythmic compound. In contrast to the block of guinea-pig myocyte I_Kr by dofetilide,³⁰ terfenadine block did not exhibit use dependence or require periodic stimulation for block onset.

Although mRNA from Kv1.5 is found in human ventricular cardiomyocytes, expression of the channel protein in the cytoplasmic membrane is low.³¹ On the basis of 4-aminopyridine sensitivity assay and expression profiles, the level of Kv1.5 may be 10-fold higher in the atrium than in the ventricle.^{31 32} The outward currents are 400-fold more sensitive to the 4-aminopyridine block in the human atrium than in the ventricle.³¹ This suggests that the contribution of Kv1.5 to ventricular repolarization is negligible compared with that of HERG. Furthermore, the likely candidate for the second component of I_K in the ventricle is I_sK,^{33 34 35} which has much slower kinetics than Kv1.5 and is weakly activated in the region of the action potential plateau (-20 to 10 mV).

The greater susceptibility of HERG to block by terfenadine combined with the low expression of Kv1.5 in the ventricle may explain the preponderance of polymorphic ventricular, as opposed to supraventricular, arrhythmias associated with terfenadine cardiotoxicity. The bell-shaped I/V of HERG spreads over a range of potentials (-40 to +40 mV) that encompasses the action potential plateau. Low doses of terfenadine therefore will introduce a regional decrease of repolarizing outward current through the primary blockade of HERG. This delimited range of potential for the action of low doses of terfenadine is ideally suited as an explanation for the acquired long QT associated with coadministration of terfenadine and ketoconazole. The results from Crumb et al¹⁹ showed that 200 nmol/L terfenadine blocked 50% of the dofetilide-sensitive current I_Kr and only 30% of I_Ks in human atrial cardiomyocytes. This small difference in the sensitivity of the channels to terfenadine block could plausibly create a change in the current-density ratio, leading to the appearance of EADs, as proposed by Zeng et al.³⁶ It remains to be determined, however, whether the results of the simulations could be applied to human ventricular cells, since, as mentioned by Zeng et al, their simulations applied to cells with a high I_Ks-to-I_Kr current-density ratio and specific blockade of I_Kr in human ventricle have been shown to induce EADs.³⁷

The properties of the dofetilide-sensitive I_Kr current in cardiomyocytes^{13 19 27} compare well with the outward HERG currents reported in this study. The K_d value found for the terfenadine block on HERG is well within the reported sensitivity of I_Kr¹⁹ and strengthen the link between I_Kr and HERG. The terfenadine K_d value of 350 nmol/L for HERG block is also physiologically relevant, since the highest clinical concentration of terfenadine in human plasma reported is in the nanomolar range (100 nmol/L).³⁸

The action of terfenadine on Kv1.5 expressed in Xenopus oocytes showed features identical to the ones reported for I_Kur in human atrial myocytes and Kv1.5 expressed in human embryonic kidney cells.^{19 26} We found an increased rate of inactivation of the Kv1.5 current in the presence of terfenadine, which suggests a fast open-state block combined with a tonic block in the resting state. The K_d for the steady state was in the micromolar range, as previously reported for extracellular application of terfenadine on cardiomyocytes and human embryonic kidney cells. These findings compare well with the known action of this antihistamine on I_Kur.^{19 26}

The time needed to reach a steady-state block was, however, markedly different for HERG than for Kv1.5, at 7 and 20 minutes, respectively. Since we used the same expression system for both channels, we exclude the possibility that differences in the diffusion of terfenadine through the cytoplasmic membrane are responsible for the gap. This is further supported by the foot portions of the dose-response curves for HERG and Kv1.5 (Figs 1 and 4), which both show a saturation of the block for concentrations >100 µmol/L. The plateau in the maximal block suggests a limited diffusion of the hydrophobic compound, which is probably due to the permeability properties of the cytoplasmic or vitelline membrane of the oocytes.

Whole-cell Kv1.5 currents expressed in human embryonic kidney cells required 5 minutes to reach steady-state levels of block,^{19 26} whereas a reversible block was reached within 2 minutes when excised macropatches were used, suggesting that the terfenadine binding site is on the cytoplasmic side of the channel. This is probably true for HERG channels also, given the saturation of the block at 100 µmol/L. The difference in the time needed for a steady-state block is more easily explained by the different affinities of the two proteins for terfenadine. Since the channels do not belong to the same family, differences in the residues forming the binding site have to be considered. Alternatively, the residues lining the access pathway may give different levels of protonation of the terfenadine molecule and therefore different affinities and binding kinetics. This pH-dependent type of affinity has been shown on I_Kur from human atrial cardiomyocytes.¹⁹ Further mutational studies on both channels should provide an answer.

In conclusion, we have shown that HERG is much more sensitive to terfenadine block than Kv1.5, the only other identified human cardiac K⁺ channel target for this drug. The buildup of terfenadine is the likely explanation for cardiotoxicity, since terfenadine carboxylate, its major metabolite, does not block either HERG or Kv1.5. This result agrees with the hypothesis that hepatic metabolism is altered by terfenadine administered concomitantly with ketoconazole. Since terfenadine carboxylate³⁹ does not block Kv1.5 or HERG potassium currents, antihistamines can be designed to be safer on the basis of this carboxylated structure in a manner that will not affect their H1 receptor antagonist properties.

	Selected Abbreviations and Acronyms

 

EAD = early afterdepolarization IK = delayed rectifier current IKr = rapid delayed rectifier current IKs = slow delayed rectifier current IKur = ultrarapid delayed rectifier K+ current IsK = slow delayed rectifier clone (minK) I/V = current-voltage, current voltage relationship QTc = QT interval corrected for heartbeat

	Acknowledgments

 
We would like to thank Drs Mark Keating and Michael Sanguinetti (University of Utah Health Sciences Center, Salt Lake City), who kindly gave us the HERG clone, and Hoeschst Marion Roussel (formerly Marion Merrell Dow) for their gift of MDL16,455. We also thank Dr Wei-Qiang Dong and Cheng Di Zuo for oocyte injections and Suzanne Davidson for expert secretarial assistance. Dr Roy was supported by a grant from the American Heart Association (Northeast Ohio Affiliate), Dr Dumaine was supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada and by the Fonds de la Recherche en Sante du Quebec, and Dr Brown was supported by NIH grant NS-23877.

Received January 25, 1996; revision received June 11, 1996; accepted June 17, 1996.

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