Hydroxyzine, a First Generation H1-Receptor Antagonist, Inhibits Human Ether-a-go-go–Related Gene (HERG) Current and Causes Syncope in a Patient With the HERG Mutation

J Pharmacol Sci 108, 462 – 471 (2008)4

Journal of Pharmacological Sciences ©2008 The Japanese Pharmacological Society

Full Paper

Hydroxyzine, a First Generation H1-Receptor Antagonist, Inhibits Human Ether-a-go-go–Related Gene (HERG) Current and Causes Syncope in a Patient With the HERG Mutation Tomoko Sakaguchi1, Hideki Itoh1, Wei-Guang Ding2, Keiko Tsuji1, Iori Nagaoka1, Yuko Oka1, Takashi Ashihara1, Makoto Ito1, Yoshihiro Yumoto3, Naoko Zenda4, Yukei Higashi4, Youichi Takeyama4, Hiroshi Matsuura2, and Minoru Horie1,* 1

Department of Cardiovascular Medicine, 2Division of Physiology, Shiga University of Medical Science, Otsu 520-2192, Japan 3 Department of Cardio-Angiology, Kitasato University School of Medicine, Sagamihara 228-8555, Japan 4 Division of Cardiology, Showa University Fujigaoka Hospital, Yokohama 227-8501, Japan Received July 4, 2008; Accepted October 14, 2008

Abstract. QT prolongation, a risk factor for arrhythmias, can result from genetic variants in one (or more) of the genes governing cardiac repolarization as well as intake of drugs known to affect a cardiac K+ channel encoded by human ether-a-go-go–related gene (HERG). In this paper, we will report a case of drug-induced long QT syndrome associated with an H1-receptor antagonist, hydroxyzine, in which a mutation was identified in the HERG gene. After taking 75 mg of hydroxyzine for several days, a 34-year-old female began to experience repetitive syncope. The deleterious effect of hydroxyzine was suspected because QTc interval shortened from 630 to 464 ms after cessation of the drug. Later on, the patient was found to harbor an A614V-HERG mutation. By using the patch-clamp technique in the heterologous expression system, we examined the functional outcome of the A614V mutation and confirmed a dominantnegative effect on HERG expression. Hydroxyzine concentration-dependently inhibited both wild-type (WT) and WT / A614V-HERG K+ currents. Half-maximum block concentrations of WT and WT / A614V-HERG K+ currents were 0.62 and 0.52 µM, respectively. Thus, accidental combination of genetic mutation and intake of hydroxyzine appeared to have led to a severe phenotype, probably, syncope due to torsade de pointes. Keywords: hydroxyzine, QT prolongation, human ether-a-go-go–related gene (HERG), potassium channel, torsade de pointes

(5 – 9). We recently experienced a case with repetitive syncope after taking a first generation H1-receptor blocker, hydroxyzine. The index patient was later found to have a heterozygous HERG mutation, A614V. As newer classes of H1-receptor blockers were shown to inhibit HERG channels, we examined whether therapeutic concentrations of hydroxyzine also affect reconstituted wild type (WT) and mutant HERG channels using a heterologous expression system with Chinese hamster ovary (CHO) cells. The A614V mutant channels were nonfunctional and showed dominant-negative suppressions on WT HERG channel expression. The mutant subunit was refractory to the intracellular trafficking and this was the major mechanism for the

Introduction A number of drugs prolonging QT interval have been shown to inhibit human ether-a-go-go–related gene (HERG) currents (1), and this fact was noted in the heterologously reconstituted HERG channels in a mammalian cell line. Terfenadine and astemizole are H1-receptor antagonists that were shown to block HERG channels (2 – 4) and have been withdrawn from the market because of reported torsade de pointes (TdP) *Corresponding author. [email protected] Published online in J-STAGE on December 5, 2008 (in advance) doi: 10.1254/jphs.08178FP

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reduced K+ conductance. The therapeutic concentration range of hydroxyzine inhibited both WT channels and those expressing equimolar WT and mutant clones (mimicking heterozygous condition) at similar halfinhibiting concentrations. These findings indicated that the first generation H1-blocker hydroxyzine also has a potential to induce QT prolongation, especially in the presence of other triggering factors such as genetic variants and hypokalemia. Materials and Methods Genomic DNA isolation and mutation analysis Genomic DNA was prepared from peripheral blood leukocytes by using QIAamp DNA Blood Midi Kits (Qiagen, Valencia, CA, USA). All exons of the HERG, KCNQ1, SCN5A, KCNE1, KCNE2, and KCNJ2 genes were amplified by PCR using intronic primer sequences. Genetic screening was performed by denaturing high performance liquid chromatography (DHPLC) using a WAVE System Model 3500 (Transgenomic, Omaha, NE, USA). Abnormal conformers were amplified by PCR and sequencing was performed on an ABI PRISM 3130 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Site-directed mutagenesis and expression, cell culture and transfection Complementary DNAs (cDNAs) for HERG (GenBank AF363636) were kind gifts from Dr. M. Sanguinetti (University of Utah, Salt Lake City, UT, USA). The cDNA was subcloned into the pRcCMV plasmid (Invitrogen, Carlsbad, CA, USA) via pBluescript II KS(+) (Stratagene, La Jolla, CA, USA) (pRcCMV / WTHERG). Site-directed mutagenesis of HERG was performed by an overlap extension PCR strategy. Nucleotide sequence analysis was performed on each variant construct before the expression study. CHO cells were maintained in Dulbecco’s modified Eagle medium and Ham’s F12 nutritional mixture (Gibco-BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Gibco-BRL) and antibiotics (100 U/ ml penicillin and 100 µg/ ml streptomycin) in a humidified incubator gassed with 5% CO2 and 95% air at 37°C. The cells were transiently transfected with either pRcCMV/ HERG (WT and / or mutant) or pIRES-CD8/ KCNQ1 + pIRES-CD8/ KCNE1 using the LipofectAMINE method according to the manufacturer’s instructions (Invitrogen). The amount of plasmids was 1 or 2 and 0.5 µg of GFP for a 35-mm dish, and only GFPpositive cells were used for the patch-clamp study.

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Electrophysiological experiments At 24 – 48 h after transfection, whole-cell patchclamp recordings were conducted at 37 ± 1°C using an EPC-8 patch-clamp amplifier (HEKA, Lambrecht, Germany). No leak subtraction was used. The normal Tyrode solution contained 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 0.33 mM NaH2PO4, 5.5 mM glucose, and 5 mM HEPES (pH adjusted to 7.4 with NaOH). The pipette solution contained 70 mM potassium aspartate, 40 mM KCl, 10 mM KH2PO4, 5 mM EGTA, 1 mM MgSO4, 3 mM Na2-ATP (Sigma Chemical Co., St. Louis, MO, USA), 0.1 mM Li2-GTP (Roche Diagnostics GmbH, Mannheim, Germany), and 5 mM HEPES (pH adjusted to 7.4 with KOH). Hydroxyzine (Sigma) was dissolved in dimethyl-sulfoxide as a stock solution at a concentration of 100 mM and was kept in the dark at −20°C. Fresh drug solutions were prepared from the stock solutions immediately before use. Whole-cell membrane current was recorded using the whole-cell configuration of the patch-clamp technique with an EPC-8 amplifier (HEKA). A coverslip with adherent CHO cells was placed on the glass bottom of a recording chamber (0.5 ml in volume) mounted on the stage of an inverted microscope (TE2000-U; Nikon, Tokyo). Pipette resistance was 3 – 5 MΩ when filled with internal solution. Currents and voltages were digitized and voltage commands were generated through an LIH-1600 AD/ DA interface (HEKA) controlled by PatchMaster software (HEKA). Current amplitude was divided by membrane capacitance (Cm) to obtain current densities (pA / pF) in each cell. The voltagedependence of current activation was determined by fitting the normalized tail current (Itail) versus test potential (Vtest) to a Boltzmann function expressed by the following equation: Itail = 1 / (1 + exp[(V0.5 − Vt) / k]), where V0.5 is the voltage at which the current is halfmaximally activated and k is the slope factor. When we analyze steady-state inactivation, the corrected steady-state inactivation curves were fitted with a Boltzmann function in the following form: I / (Imax − Imin) = 1 / {1 + exp[(Vt − V0.5) / k]} + Imin, where I is the amplitude of inactivating current corrected for deactivation, Imax is the maximum of I, Imin is the minimum of I, Vt is the prepulse of test potential, V0.5 is the voltage at which I is half of Imax, and k is the slope factor. Data analyses Numerical data are presented as mean ± S.E.M. Student’s t-test was used to compare the data between different groups for electrophysiological measurements, and differences were considered significant at P<0.05.

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Fig. 1.

Twelve-lead ECG traces. A: ECG the next day after syncope. B: ECG three weeks after discontinuing hydroxyzine.

Cell preparation and confocal imaging For confocal microscopy experiments, green fluorescent protein (GFP) was tagged to the C terminus of WT or A614V HERG using EGFP-N1 vector (Clontech, Mountain View, CA, USA). HEK293 cells were plated on 35-mm glass-bottom culture dishes and maintained for 12 – 24 h in Dulbecco’s modified Eagle’s medium containing 10% FBS. Lipofection was performed with Lipofectamine reagent (Invitrogen). At 48 h after transfection, HEK293 cells were incubated for 15 – 18 h with 50 µM cycloheximide. The localization of GFP-tagged proteins was detected using an LSM 510-META laserscanning microscope (Carl Zeiss, Thornwood, NY, USA) with a 63 × 1.40 numerical aperture oil-immersion objective (10). GFP was excited using a 488-nm line of an argon laser and signals were collected through a 500 – 530 band-pass filter. Results Case presentation For the treatment of chronic pruligo, the 34-year-old woman began to take hydroxyzine (75 mg / day) and 3 days later, she experienced repetitive syncope. On admission, her ECG showed a marked QT prolongation (640 ms, QTc = 630 ms; Fig. 1A), and her syncope was suspected to be due to TdP. She had no particular family

history, but once had a pre-syncopal attack of unknown origin several years ago. Immediately after admission, hydroxyzine intake was stopped, and she was free of syncope since then. QT intervals gradually shortened to 460 ms (QTc = 464 ms) three weeks after the episode (Fig. 1B). It was therefore evident that the drug induced an extreme prolongation of QT interval. Because her QT interval remained slightly prolonged after the cessation of hydroxyzine, we conducted genetic testing and identified a heterozygous missense HERG mutation, A614V, causing a substitution of alanine at codon 614 to valine (Fig. 2: A – C). Panel A of Fig. 2 depicts the original results of screening by DHPLC, and panel B shows the DNA sequence data. Panel C shows her family tree; her two children were negative for this mutation. There was a heterozygous substitution of C to T at position 1841 in exon 7, which resulted in A614V. The mutation is located in the pore region (Fig. 2D) and has been known as a hot spot for the congenital LQTS (11 – 13). Biophysical assay of wild type and mutant HERG channel function In order to clarify the mechanism underlying the marked QT prolongation and syncope presumably due to TdP after intake of hydroxyzine, we conducted electrophysiological experiments by using CHO cells express-

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Fig. 2. Genetic testing revealed that the index patient had a missense HERG mutation. A: DHPLC analysis of PCR products of family members: from top to bottom, a: control, b: proband, c: her son, d: her daughter. The control, her son, and her daughter displayed one normal peak pattern, but the proband, in contrast, showed a twopeak pattern. B: Sequence identification of a C to T transition of one HERG allele in the proband (both alleles shown), causing replacement of alanine 614 by valine (A614V). C: Partial pedigree of the family (squares indicate men and circles, women). Filled symbol depicts mutation carrier and symbols with N, non-carriers; open symbol, family member without available findings. D: Schematic representation of the predicted topology of the HERG protein and the location of A614V. C = C-terminal domain and N = N-terminal domain.

ing either WT or mutant HERG channel. Figure 3A shows four sets of representative current traces recorded from CHO cells transfected with WT (2 and 1 µg, panels a and b), A614V alone (2 µg, panel c), or WT / A614V (1 µg each, panel d). Depolarization pulses applied from a holding potential of −80 mV to various test potentials elicited time-dependent outward K+ currents, followed by the decaying outward tail current upon return to −60 mV in cells expressing WT HERG (Fig. 3A, panels a and b). In contrast, cells expressing A614VHERG alone did not produce any measurable currents (Fig. 3A, panel c). When expressed with WT and A614V cDNA at 1:1 ratio, cells displayed significantly smaller outward currents compared with that of WT 1 µg (Fig. 3A, panel d). As illustrated in Fig. 3B, the HERG-related K+ currents were activated at potentials positive to −40 mV. The amplitude of outward current was increased with depolarization up to 0 mV, but was then progressively decreased with further depolarization, which is typical for an inward rectifying property of hERG (14). Therefore, when values for the maximum peak of steady state current during depolarizing pulses were plotted as a function of test potentials (Fig. 3B), the I-V curve showed a bell-shaped relation. Compared to the case of WT (1 µg, open squares, Fig. 3B), WT / A614V HERG channel (filled diamonds) displayed nearly 70% reduction in current density.

Peak tail currents elicited on repolarization to −60 mV from various test potentials were measured and then normalized by the cell capacitance in multiple cells. These values were plotted as a function of test potentials (Fig. 3C) and fitted with a Boltzmann equation, yielding half maximum activation potential (V0.5) and slope factor (k) (Table 1). Maximum peak current densities measured at +50 mV in WT-HERG channels were 43.2 ± 4.0 and 29.4 ± 3.4 pA/ pF (2 and 1 µg, respectively). In contrast, those in the WT / A614V-HERG channels were 7.9 ± 1.1 pA / pF and were significantly smaller (approximately one fourth) than those of the 1 µg WT channels. However, co-expression of A614V did not significantly alter V0.5 and k values (Table 1). Thus, A614V appeared to cause a dominant-negative suppression on WT-HERG channel subunits without affecting the kinetic parameters. We examined steady-state inactivation using a dualpulse protocol (Fig. 3D). There was no significant change in its voltage dependence between WT and WT / A614V. V0.5 and the slope factor of steady-state inactivation were not different between WT and WT / A614V [V0.5: −51.0 ± 2.8 for WT (n = 6), −54.4 ± 2.3 for WT / A614V (n = 7); slope factor: −27.4 ± 1.6 for WT, −26.3 ± 5.1 for WT/ A614V].

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Fig. 3. Functional assays of A614V HERG channel in CHO cells. A: Representative current traces recorded from cells expressing the constructs indicated above each graph. Currents were elicited from a holding potential of −80 mV, by depolarizing pulses from −50 to +50 mV (with a 10-mV step increment) and subsequent repolarization to −60 mV. B: Steady state current– voltage relationships for WT-HERG (2 μg; open circles, n = 26), WT-HERG (1 μg; open squares, n = 22), A614V /WT-HERG (filled diamonds, n = 21), and A614V-HERG (filled triangles, n = 8). C: Tail current–voltage relations for WTHERG (1 μg; open squares, n = 22), A614V /WTHERG (filled diamonds, n = 21), and A614VHERG (filled triangles, n = 8). **P<0.01 vs WTHERG (1 μg). D: Normalized steady-state inactivation curves of expressed currents in CHO cells transfected with WT-HERG (open circles, n = 6) or A614V /WT-HERG (filled diamonds, n = 7). To examine the steady-state inactivation, conditioning pulses between −120 and +20 mV in 10mV increments for 10 ms were applied after a depolarizing pulse to +20 mV for 900 ms, followed by a common test pulse to +20 mV. The voltage protocol is illustrated in the inset. Peak current amplitudes during test pulses were plotted as a function of the previous potential. Normalized steady-state inactivation–test potential relations were fitted to a Boltzmann function. Table 1.

Parameters of activation in currents expressing WT-HERG and/or A614V-HERG WT (2 μg) (n = 26)

Peak tail current (pA / pF) V0.5 (mV) k (mV)

WT (1 μg) (n = 22)

WT (1 μg) / A614V (1 μg) (n = 20)

43.2 ± 4.0

29.4 ± 3.4

−20.6 ± 1.2

−20.8 ± 1.0

−22.7 ± 1.0

7.9 ± 1.1**

7.6 ± 0.3

7.8 ± 0.2

7.1 ± 0.3

A614V (2 μg) (n = 10) 0.6 ± 0.5**

**P<0.01 vs WT (1 μg).

Hydroxyzine inhibited both WT and WT /A614V HERG currents In the next series of experiments, we examined whether hydroxyzine exerts inhibitory effects on reconstituted HERG currents to explain the drug-induced cardiac event in our index patient. Figure 4A shows a representative experiment to examine the effect of hydroxyzine on WT-HERG current. Current traces were measured before (left) and after (right) extracellular application of 1 µM hydroxyzine. Time-dependent

outward current during depolarizing pulses as well as tail currents elicited on return to −60 mV were reduced by hydroxyzine. In Fig. 4, B and C, tail current and normalized tail current densities are plotted against test potentials (open circles: control and filled circles: 1 µM hydroxyzine). As depicted in Fig. 4B, hydroxyzine significantly decreased WT-HERG currents (P<0.01 vs WT-HERG, Table 2). Hydroxyzine also modified the voltage-dependence of activation of the WT-HERG channels and produced a significant shift of V0.5 toward

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Fig. 4. Hydroxyzine block of WT-HERG K+ currents. Representative current traces recorded in CHO cells expressing WT-HERG in control condition (a) and in the presence of hydroxyzine (1 μM) (b). Tail current–voltage relations for the control condition (open circles, n = 16) and in the presence of hydroxyzine (1 μM) (filled circles, n = 16). **P<0.01 vs WT-HERG. Normalized activation curves.

Table 2.

Parameters of activation in currents in the presence of hydroxyzine (1 μM)

WT (n = 16) Peak tail current (pA / pF) V0.5 (mV) k (mV)

WT + hydroxyzine (1 μM) (n = 16)

WT/A614V (n = 13)

WT/ A614V + hydroxyzine (1 μM) (n = 13)

45.1 ± 4.0

15.3 ± 3.3**

6.0 ± 0.9

2.1 ± 0.6**

−19.9 ± 0.9

−26.7 ± 1.1**

−20.4 ± 1.2

−28.6 ± 2.4**

7.6 ± 0.3

7.9 ± 0.4

7.2 ± 0.3

6.1 ± 0.7

**P<0.01 vs control (absence of drug).

more negative potentials (Fig. 4C, Table 2). Similarly, HERG currents reconstituted by the co-transfection of WT and A614V were also significantly inhibited by hydroxyzine (Table 2 and see also Fig. 5A). The drug sensitivity of HERG channel currents were extensively assessed by applying various concentrations of extracellular hydroxyzine under the whole-cell condition by means of the action potential command method (Fig. 5A). In the inset, the voltage-clamp protocol is shown, and the pulses with action potential configuration are given at 1 Hz. As shown in panel a of Fig. 5A, the compound (0.5 and 1 µM) inhibited WT (1 µg) HERG currents in a concentration-dependent manner (red trace: control, blue trace: 0.5 µM, and dark blue trace: 1 µM hydroxyzine). Percent inhibitions of peak currents during action

potential command waveforms were measured at six different concentrations of hydroxyzine and are plotted as a function of its concentrations (Fig. 5B, open circles). Smooth curves were drawn by fitting them to a Hill equation, yielding hydroxyzine a half maximum inhibitory concentration (IC50) of 0.62 µM and Hill coefficient of 0.99. We examined hydroxyzine concentration–inhibition relation for WT / A614V (1 µg each) HERG currents as conducted on WT currents. As shown in panel b of Fig. 5A, WT/ A614V currents were also inhibited by micromolar concentrations of hydroxyzine. Its concentration–inhibition curve (Fig. 5B, filled circles) almost overlapped that observed in regard to WT-HERG currents. Fitting to a Hill equation yielded the IC50 of 0.52 µM and the Hill coefficient of 1.01. These values

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Fig. 5. Concentration-dependent block of both WT and WT/A614V HERG by hydroxyzine. Representative current traces recorded in CHO cells expressing WT HERG (a) and WT/A614V HERG (b) in control condition and in the presence of increasing hydroxyzine concentrations. An action potential wave form recorded from a cat ventricular myocyte was used as the command voltage to clamp. HERG currents are expressed as a normalized E-4031-sensitive current (control current – residual current in 3 μM E-4031). Dose– response curve for hydroxyzine to block WT and WT/A614V HERG K+ current. Current was normalized to the control value and expressed as a function of the drug concentration. Solid line, fit of experimental data to the Hill equation: y = a + {d / [1 + (x/c)nH]}, where x is the drug concentration and nH is the Hill coefficient. IC50 value was calculated as the drug concentration for which y = 50%.

Fig. 6. Subcellular distribution of WT, A614V and WT/ A614V in HEK293 cells. Representative microscopic images of HEK293 cells expressing GFP-tagged WT HERG (three upper panels), GFP-tagged A614V mutant (middle three), and GFPtagged WT HERG co-transfected with non-tagged A614V mutant (lower three). The three panels in the left column show contrast microscopic images and the middle 3 panels, corresponding confocal microscopic images. The three panels in the right column display magnified images of yellow-boxed areas in the left and middle column panels. Yellow arrows on the images indicate the margin between the surface membrane and extracellular space.

Hydroxyzine Block of HERG Channel

were not statistically different from those obtained for WT-HERG currents. We also examined the effect of hydroxyzine on KCNQ1/ KCNE1 currents reconstituted in CHO cells. Hydroxyzine did not inhibit KCNQ1 / KCNE1 currents at 1 μM concentration, and 10 μM of the drug only inhibited the KCNQ1 / KCNE1 currents by 9 ± 2.9% (n = 8) (data not shown). A614V HERG is a trafficking-deficient mutation The results of electrophysiological experiments showed that hydroxyzine exerted a similar inhibitory effect on both WT and WT / A614V channel currents (Fig. 5) and that both types of channels showed a similar voltage-dependent activation process (Fig. 3). Using a Western blot analysis, Anderson et al. (15) demonstrated that A614V is a trafficking-deficient mutation. Collectively, the A614V mutant subunit appeared refractory to the intracellular transport, and all the expressed channels were WT. In order to examine the intracellular transport of WT and mutant HERG channels in more detail, we finally conducted confocal imaging experiments using GFP-tagged clones. Whole-cell patch-clamp experiments confirmed that the GFP-tagged WT clone (WT-GFP) could successfully generate a similar size of typical HERG currents in CHO cells (data not shown). The left column of Fig. 6 depicts three confocal images in HEK293 cells expressing GFP-tagged WT (upper), GFP-tagged A614V (A614V-GFP) alone (middle), and GFP-tagged WT with non-tagged A614V (WT-GFP / A614V) (lower). The panels in the left column show corresponding images taken by a phase-contrast microscope. The regions of interest are indicated by small squares in the merged and expanded images (right) of green fluorescence (middle) and light transmission (left). WT-HERG channels were amply transported to the cell surface membrane; in contrast, A614V channels failed to attain a position on the membrane surface (indicated by yellow arrows). GFP signal obtained from WT-HERG in cells co-transfected with non-tagged A614V (right lower panel) could be detected but only scantily in the cell surface membrane. Thus, the findings confirmed the electrophysiological results, suggesting that A614V may trap WT-HERG protein and thereby hinder its cell surface expression. Discussion In the present study, submicromolar concentrations of (<1 μM) hydroxyzine hydrochloride, a first generation H1-receptor blocker, were found to inhibit reconstituted HERG K+ currents. The drug is a classical antihistamine with antiallergic, antimuscarinic, and sedative actions

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and has also cardiac adverse effects such as abnormal ventricular repolarization (16). Wang et al. (17) reported that the drug prolonged the QT interval in an isolated feline heart, and Taglialatela et al. examined the effects of first-generation antihistamines, including hydroxyzine, on human neuroblastoma cells and Xenopus oocytes expressing HERG channel (18). Other first generation H1-receptor blockers such as chlorpheniramine, pyrilamine, and diphenhydamine were also indicated to block the rapidly activating component of the delayed rectifier potassium current, IKr (19 – 21). More recently, newer H1-receptor antagonists, terfenadine and astemizole, were withdrawn from the market because of TdP occurrence (5 – 9). These antagonists have been shown to inhibit cardiac IKr currents in a concentrationdependent manner with IC50 values of 0.35 – 0.45 μM (3, 21). The hydroxyzine IC50 for HERG currents (0.62 μM) in our study is close to its reported maximum serum concentration in humans (0.16 μM after a single oral dose of 0.7 mg / kg hydroxyzine), and the drug has a very long washout time (t0.5 = 20 h) (22, 23). We should therefore pay attention to its potential adverse action on QT intervals. Taglialatela et al. (18) reported that hydroxyzine’s IC50 for HERG in oocytes was 10.7 μM, which is far higher than normal therapeutic concentrations. The differences in IC50 may reflect different experimental conditions, and we think that our data are more clinically relevant. Both cardiac and non-cardiac agents are known to cause acquired drug-induced LQTS (24) through the inhibition of HERG channel currents (4). Hydroxyzine does not inhibit IKs at its therapeutic concentration range. Marked QT prolongation and repetitive syncope in our index patient were therefore associated with hydroxyzine-induced inhibition of HERG currents. However, the patient had another potential for the lifethreatening arrhythmia: heterozygous mutation of the HERG gene. The unexpected combination of two risk factors appeared to cause a severe phenotype in this specific case. On functional assessment, the gene variant was found to reduce native HERG currents to onefourth, and hydroxyzine still blocked WT / A614V HERG currents with a similar potency. Thus, even lower serum concentration of hydroxzyine would have produced considerable QT prolongation and presumably TdP, which may be a cause of the patient’s syncope. In the functional study, the A614V HERG mutation identified in our patient showed dominant negative effects on WT-HERG channels without affecting parameters of activation and inactivation. Using Western blot analyses, Andersen et al. (15) also reported that A614V is a trafficking-deficient mutation. In accordance with this report, we showed that A614V caused

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deficiency in intracellular transport (protein trafficking) to the cell surface membrane, by using the confocal imaging method. Our methods could confirm their results and directly show the location of targeted proteins and added more findings on the transporting process of these proteins, especially when co-expressed. Therefore, A614V subunit incorporation into normal assembly appeared to prevent its healthy traffic to the plasma membrane. Using a heterologous expression system with Xenopus oocytes, Nakajima et al. (25) demonstrated that A614V suppressed HERG current in a dominant-negative manner through the enhancement of inward rectification resulting from the shift in voltage dependence of HERG inactivation. The inconsistency of the results about the voltage dependence of the steadystate inactivation may due to the difference of the cells used for experiments. Impaired trafficking of HERG protein is increasingly recognized as a leading cause of type 2 LQTS. For several trafficking-deficient HERG channels, incubation of cells at reduced culture temperatures or with HERG blockers has been shown to rescue the deficient transport (15). Concerning the A614V-HERG channel, both thapsigargin and E4031 failed to improve the protein processing (15). We also tested whether hydroxyzine rescued the channel trafficking, but no recovery was observed (data not shown). In conclusion, QT-prolonging drugs usually require the presence of multiple risk factors to manifest lifethreatening arrhythmias (1). In our patient, both genetic background and female sex are definite risk factors to trigger the QT prolongation. In addition, hydroxyzine intake produced a “double hit”, leading to a reduction in cardiac “repolarization reserve” (26) and a severe phenotype, presumably TdP. Thus, genetic screening is of clinical importance not only for familial LQTS patients but also for patients with drug-induced QT prolongation. When possible genetic background is identified, the genetic analysis is especially necessary for the proband’s family members to avoid unexpected drug-induced adverse actions. Based on our electropharmacological study, hydroxyzine should be avoided for patients with prolonged QT intervals and be included in the list of drugs (http:// www.torsades. org / medicalpros / drug-lists / drug-lists.cfm) that prolong the QT interval and / or induce torsades de pointes ventricular arrhythmia. Acknowledgment We thank Ms. A. Ikeda for her excellent technical support. This study was supported by the grants from Ministry of Education, Culture, Sports, Science, and

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