Effect of Terfenadine and Pentamidine on the hERG Channel and Its Intracellular Trafficking: Combined Analysis with Automated Voltage Clamp and Confocal Microscopy

Abstract

The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.

Treatment with certain cardiovascular and non-cardiovascular drugs is known to induce QT prolongation and serious ventricular arrhythmia including torsades de pointes.^1–3) Drugs such as terfenadine⁴⁾ and cisapride⁵⁾ were withdrawn from the market because of their arrhythmogenic risk. Nowadays, the assessment of the arrhythmogenic risks incurred with noncardiovascular therapeutic agents is inevitable. One of the standard methods to evaluate the tendency of drugs to induce QT prolongation and ventricular arrhythmia is examination of drug effects on the human ether-a-go-go related gene (hERG) channel, which encodes the rapid component of the human delayed rectifier potassium current (I_Kr).⁶⁾ The hERG channel is the overwhelmingly most common target of torsades de pointes-inducing drugs. Heterologously expressed hERG channels lack the ancillary subunit MiRP1, and is thus not identical to the native channel underlying I_Kr.⁷⁾ Nevertheless, assessment of hERG-blocking activity has been proven to be valid for the risk evaluation of a majority of drugs.^8–10) However, there are some drugs which have no acute effect on the hERG channel current, but induce QT prolongation and ventricular arrhythmia such as pentamidine,^11–13) probucol^14,15) and cardiac glycosides.¹⁶⁾ These drugs have been reported to inhibit the intracellular trafficking of the hERG channel to the cell membrane. Long term application of such drugs can cause a decrease in the density of the hERG channel on the myocardial cell membrane and lead to QT prolongation. Thus, evaluation of arrhythmogenic risk should include hERG channel inhibition both at the level of channel current and channel trafficking.^17–19)

The automated patch clamp system has been widely used as an efficient assay system for drug effects on various ion channels including the hERG channel.²⁰⁾ Its high throughput makes it suitable for the early-stage evaluation of test compound on the hERG channel current. It would also be useful for the evaluation of drug effects on hERG channel trafficking provided that the drug could be washed out after long-term application to hERG expressing cells. Another method to evaluate drug effects on hERG channel trafficking is confocal microscopy.²¹⁾ By using green fluorescent protein (GFP)-labeled hERG channels, the intracellular distribution of the channel can be visualized and it would be able to evaluate the fraction of the channel present on the cell membrane provided that a quantitative and practical analyzing method is developed.

In the present study, we intended to clarify whether combined use of automated voltage clamp and confocal microscopy is an efficient and practical method for the evaluation drug effects on the hERG channel current and trafficking. We examined the effects of terfenadine and pentamidine, drugs which are considered to inhibit hERG channel current and trafficking, respectively. Our results suggested that the combination of these two methods is indeed beneficial.

MATERIALS AND METHODS

Preparation of HEK293 Cells Expressing the hERG Channel-GFP Fusion Protein

cDNA fragments encoding hERG were amplified by polymerase chain reaction from a human heart cDNA library (TaKaRa Bio Inc., Seta, Japan) according to the published hERG cDNA sequence (GenBank accession number U04270)²²⁾ and assembled with standard ligation techniques as described in our previous report.²³⁾ The full-length hERG cDNA was inserted into the vector pAcGFP1-N1 (Clontech, Mountain View, CA, U.S.A.), resulting in an expression vector encoding a hERG channel with GFP fused on the C-terminus (hERG-GFP). The vector encoding hERG-GFP was introduced into cultured HEK293 cells by lipofection (FuGENE HD, Fitchburg, WI, U.S.A.), and stable transformants were obtained by culturing in the presence of 400 µg/mL G418 (Geneticin, Life Technologies, Carlsbad, CA, U.S.A.), a neomycin analog. Transformed HEK293 cell clones were observed at an excitation wavelength of 488 nm and hERG channel currents were measured as described below. Among the GFP fluorescence-positive clones, a clone with a hERG current amplitude of about 1.0 nA was chosen for analysis. The cells were plated on collagen-I coated dishes 48 to 72 h before analysis.

Electrophysiological Recording of Expressed hERG-GFP Current

The cells were detached from the dishes with Accutase (Innovative Cell Technologies, Inc., San Diego, CA, U.S.A.) and re-suspended in the external solution described below. In the case of cells chronically treated with drugs, the cells were washed with saline (5 min×3 times) before detachment, and the subsequent procedures were performed in the absence of drugs. Whole-cell voltage clamp experiments were performed with the Patchliner planar patch clamp system (Nanion Technologies, Munich, Germany). The Patchliner is an automated patch clamp device, which utilizes planar patch clamp chips made from borosilicate glass for the recordings.²⁰⁾ The center of the glass chip has approximately 1 µm-sized aperture, onto which the cell is positioned automatically by suction. The external solution was of the following composition: 140 mM sodium chloride (NaCl), 4 mM potassium chloride (KCl), 2 mM CaCl₂, 1 mM MgCl₂, 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), 5 mM D-glucose (pH 7.4 with sodium hydroxide (NaOH)). The internal solution was of the following composition: 50 mM KCl, 10 mM NaCl, 60 mM K-fluoride, 10 mM HEPES, 20 mM ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (pH 7.2 with potassium hydroxide (KOH)).²⁴⁾ The glass chips with a resistance of approximately 2–3 MΩ were used for recordings. The hERG-GFP tail current was activated every 15 s throughout the experiment by a depolarizing voltage clamp pulse to +20 mV for 1500 ms from a holding membrane potential of −80 mV, followed by a pulse to −50 mV for 1500 ms to record the tail current. Drugs were applied cumulatively, and the current was measured before and 3 min after the drug application of each concentration. Data recording and analysis were performed with a voltage clamp amplifier EPC10 Quadro (HEKA Electronics, Lambrecht, Germany), and softwares PatchMaster (HEKA Electronics) and Igor (WaveMetrics, Lake Oswego, OR, U.S.A.).

Confocal Microscopic Analysis of hERG-GFP Channel Localization

Two-dimensional imaging of HEK293 cells expressing hERG-GFP was performed with a confocal microscope LSM510 (Carl Zeiss, Oberkochen, Germany), with procedures similar to those in our previous reports.^25–28) Coverslips with cells were placed in a chamber on the stage of the inverted microscope and perfused with HEPES-Tyrode solution of the following composition (mM): NaCl 143, KCl 5.4, MgCl₂ 1.8, CaCl₂ 2, NaH₂PO₄ 0.33, glucose 5.5 and HEPES 20. The solution was gassed with 100% O₂. Experiments were performed at 37°C. For the detection of hERG-GFP channels, the cells were excited at 488 nm by an Ar⁺ laser. For the determination of the cell membrane region, the cells were stained with the membrane probe PKH26 and excited at 543 nm by a HeNe laser. The emission were detected by photomultipliers and organized into two-dimensional images. The objective used was Plan-Apocromat 63x/1.4 oil immersion. Horizontal (x–y) planes at half cellular height level were scanned in 512×512 pixels. The fraction of hERG-GFP fluorescence on the cell membrane was calculated by dividing the hERG-GFP fluorescence on the cell membrane region by the total cellular hERG-GFP fluorescence. The validity of this method was confirmed in control experiments with GFP unlinked to the hERG channel; the membrane fraction of such channels was less than 5%.

Drugs and Chemicals

Terfenadine, pentamidine and PKH26 were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). All other chemicals were commercial products of the highest available grade of quality.

RESULTS

In HEK293 cells expressing the hERG-GFP channel, depolarization to membrane potentials more positive than −50 mV induced voltage- and time-dependent outward currents (Fig. 1Aa). A time dependent decrease in outward current was observed at potentials more positive than 0 mV resulting in a bell-shaped current–voltage relationship at steady state (Fig. 1Ab). On repolarization to −50 mV, outward tail currents were observed (Fig. 1A).

Fig. 1. Typical Current Traces for the hERG-GFP Channel Current and the Effects of Terfenadine and Pentamidine

A: Typical current traces (a) and summarized current–voltage relationships (b) for the maximum outward current on depolarization to various voltages (closed circles) and the tail current amplitude on repolarization to −50 mV (open circles). Symbols and bars represent the mean±S.E.M. from 11 experiments. B: Typical current traces on depolarization to +20 mV and the following repolarization to −50 mV showing the acute (a, b) and chronic (c, d) effects of 30 nM terfenadine (a, c) and 1 µM pentamidine (b, d). Open and closed circles in a and b indicate current traces obtained in the absence and presence of drugs, respectively. Currents traces in c and d were obtained in cells chronically treated with drugs for 48 h in the culture medium and thoroughly washed before the measurement.

The acute blocking effect of drugs on the hERG-GFP channel was evaluated through measurement of the peak outward tail current on repolarization to −50 mV in the presence of the drug. Terfenadine concentration-dependently reduced the tail current amplitude (Figs. 1Ba, 2A); the concentration for 50% inhibition of the current (absolute IC₅₀ of terfenadine) was 30.6±1.8 nM (n=8). Pentamidine had no effect on the tail current amplitude at concentrations up to 3 µM; at 10 µM, pentamidine reduced the current amplitude by about 10% (Figs. 1Bb, 2B).

Fig. 2. Summarized Results for the Effects of Terfenadine (A) and Pentamidine (B)

Closed circles: Acute blocking effects on the hERG-GFP channel current were evaluated by voltage clamp. The peak outward tail current amplitude on repolarization to −50 mV after a depolarizing pulse to +20 mV in the presence of each concentration drugs expressed as a percentage of the amplitude in the absence of the drug. Symbols and bars represent the mean±S.E.M. from 7 to 8 experiments. Open circles: Effect on hERG-GFP channel trafficking evaluated by current measurement after chronic treatment in the culture medium and washout. The peak outward tail current density on repolarization to −50 mV after a depolarizing pulse to +20 mV in cells treated with each concentration of drugs was expressed as a percentage of that in untreated cells. Symbols and bars represent the mean±S.E.M. from 12 cells. Closed triangles: Effect on hERG-GFP channel trafficking evaluated by confocal microscopy. The fraction of hERG-GFP fluorescence on the cell membrane in cells treated with each concentration of drugs expressed as a percentage of the value in untreated cells. Symbols and bars represent the mean±S.E.M. from 10 cells. Asterisks indicate significant difference with corresponding values in the absence of drugs or in untreated cells as evaluated by one-way ANOVA followed by Dunnett’s test for multiple comparisons.

Electrophysiological evaluation of the chronic effect of drugs to inhibit trafficking of the hERG-GFP channel to the cell membrane was performed by application of drugs to the culture medium for 48 h and measurement of the current after washout. Terfenadine had no significant effect on the tail current amplitude at concentrations up to 30 nM; at 100 nM, terfenadine reduced the tail current amplitude by about 30% (Figs. 1Bc, 2A). Pentamidine reduced the tail current amplitude concentration-dependently (Figs. 1Bd, 2B); the concentration for 50% inhibition of the current (absolute IC₅₀ of pentamidine) was 2.84 µM (n=12).

Confocal microscopic evaluation of the chronic effect of drugs to inhibit trafficking of the hERG-GFP channel to the cell membrane was performed by application of drugs to the culture medium and measurement of the fraction of the fluorescence on the cell membrane. Terfenadine had no significant effect on the membrane fraction at concentrations up to 100 nM (Figs. 2A, 3B). Pentamidine reduced the membrane fraction concentration-dependently (Figs. 2B, 3C); concentration to reduce the membrane fraction by 50% (absolute IC₅₀ of pentamidine) was 3.71 µM (n=10).

Fig. 3. Typical Confocal Images of HEK-293 Cell Expressing hERG-GFP and the Chronic Effect of Drugs

Cells were cultured in the absence (A) or presence of 30 nM terfenadine (B) or 1 µM pentamidine (C) for 48 h. The hERG-GFP fluorescence is shown in green and the cell membrane labeled with PKH26 in red (a). The hERG-GFP labeling on the cell surface is shown in white (b). Note that hERG-GFP labeling on the cell surface was decreased in the pentamidine-treated cell (Cb).

DISCUSSION

The hERG-GFP channel current expressed in HEK293 cells had voltage-dependent activation and inactivation properties similar to those of the wild type hERG channel current^10,23) (Fig. 1A). The IC₅₀ values for the acute blocking effect of terfenadine on hERG-GFP channels (Fig. 2) were also about the same as those reported for wild type hERG channels. Quantitative confocal imaging analysis revealed that about 20% of the total cellular hERG-GFP channels were in the cell membrane region. These results confirm that the hERG-GFP channels are well incorporated into the cell membrane and retain the electrophysiological and pharmacological properties of the wild type hERG channel. Thus, the hERG-GFP channel expressed in HEK293 cells can be used for studies on drug effects on hERG channel activity and intracellular trafficking. Addition of EGFP to the amino terminus of hERG (EGFP-hERG) was reported to result in changes in gating kinetics and trafficking of the hERG channel, while no such effects were observed when EGFP was added to the carboxyl terminus (hERG-EGFP).²¹⁾

Concerning the intracellular trafficking of hERG channels, pentamidine was clearly demonstrated to have potent inhibitory effects both by electrophysiological analysis and by confocal microscopy. The IC₅₀ values obtained by these two methods were almost identical suggesting the quantitative accuracy of the two methods. IC₅₀ values of pentamidine for channel trafficking and maturation were reported to be 5.1 µM and 7.8 µM, respectively, as determined by membrane current and Western blotting analyses.¹²⁾ Thus, pentamidine reduced the hERG channel current through inhibition of channel trafficking with virtually no direct blocking effect on the channel current. On the other hand, terfenadine had no significant effect on hERG channel trafficking despite its acute blocking effect on the channel current. The apparent inhibitory effect on hERG channel trafficking at 100 nM could be attributed to the residual acute blocking effect on the channel current because of less efficient washout. Thus, it was demonstrated that terfenadine and pentamidine, the drugs which produce QT prolongation through inhibition of the hERG channel current, act through different mechanisms; terfenadine directly blocks the channel and pentamidine inhibits channel trafficking.

The electrophysiological analysis and confocal microscopy, as used in the present study, have complementary characteristics concerning the evaluation of drug effects on the hERG channel current. The confocal microscopic analysis enables evaluation of drug effects without being obscured by blocking action on the channel current. However, as the theoretical resolution limit of the confocal microscope is about 0.2 µm, the hERG-GFP channels present just beneath the cell membrane can not be distinguished from those incorporated in the cell membrane. Voltage clamp analysis of hERG channel currents, which detects only the channels that are incorporated into the cell membrane, is free from such limitation. On the other hand, the acute blocking effect of drugs can not be distinguished from trafficking inhibition by voltage clamp analysis when drug washout is not efficient enough, as was the case with terfenadine. Thus, combined use of voltage clamp and confocal microscopy appears to be a powerful and practical method for the evaluation of drug effects on the function and trafficking of hERG channel.

REFERENCES

• 1) Tamargo J. Drug-induced torsade de pointes: from molecular biology to bedside. Jpn. J. Pharmacol., 83, 1–19 (2000).
• 2) Hashimoto K. Arrhythmia models for drug research: classification of antiarrhythmic drugs. J. Pharmacol. Sci., 103, 333–346 (2007).
• 3) Fermini B, Fossa AA. The impact of drug-induced QT interval prolongation on drug discovery and development. Nat. Rev. Drug Discov., 2, 439–447 (2003).
• 4) Salata JJ, Jurkiewicz N, Wallace A, Stupienski R 3rd, Guinosso P Jr, Lynch J Jr. Cardiac electrophysiological actions of the histamine H1-receptor antagonists astemizole and terfenadine compared with chlorpheniramine and pyrilamine. Circ. Res., 76, 110–119 (1995).
• 5) Bran S, Murray W, Hirsch I, Palmer J. Long QT syndrome during high-dose cisapride. Arch. Intern. Med., 155, 765–768 (1995).
• 6) Sanguinetti MC, Jiang C, Curran ME, Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the I_Kr potassium channel. Cell, 81, 299–307 (1995).
• 7) Abbott GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating MT, Goldstein SA. MiRP1 Forms I_Kr potassium channels with HERG and is associated with cardiac arrhythmia. Cell, 97, 175–187 (1999).
• 8) Farkas AS, Nattel S. Minimizing repolarization-related proarrhythmic risk in drug development and clinical practice. Drugs, 70, 573–603 (2010).
• 9) Witchel HJ. Drug-induced hERG block and long QT syndrome. Cardiovas. Ther, 29, 251–259 (2011).
• 10) Takahara A, Fujiwara K, Ohtsuki A, Oka T, Namekata I, Tanaka H. Effects of the antitussive drug cloperastine on ventricular repolarization in halothane-anesthetized guinea pigs. J. Pharmacol. Sci., 120, 165–175 (2012).
• 11) Cordes JS, Sun Z, Lloyd DB, Bradley JA, Opsahl AC, Tengowski MW, Chen X, Zhou J. Pentamidine reduces hERG expression to prolong the QT interval. Br. J. Pharmacol., 145, 15–23 (2005).
• 12) Kuryshev YA, Ficker E, Wang L, Hawryluk P, Dennis AT, Wible BA, Brown AM, Kang J, Chen XL, Sawamura K, Reynolds W, Rampe D. Rampe. Pentamidine-induced long QT syndrome and block of hERG trafficking. J. Pharmacol. Exp. Ther., 312, 316–323 (2005).
• 13) Yokoyama H, Nakamura Y, Iwasaki H, Nagayama Y, Hoshiai K, Mitsumori Y, Sugiyama A. Effects of acute intravenous administration of pentamidine, a typical hERG-trafficking inhibitor, on the cardiac repolarization process of halothane-anesthetized dogs. J. Pharmacol. Sci., 110, 476–482 (2009).
• 14) Elharrar V, Watanabe AM, Molello J, Besch HR Jr, Zipes DP. Adrenergically mediated ventricular fibrillation in probucol-treated dogs: roles of alpha and beta adrenergic receptors. Pacing Clin. Electrophysiol., 2, 435–443 (1979).
• 15) Guo J, Massaeli H, Li W, Xu J, Luo T, Shaw J, Kirshenbaum LA, Zhang S. Identification of I_Kr and its trafficking disruption induced by probucol in cultured neonatal rat cardiomyocytes. J. Pharmacol. Exp. Ther., 321, 911–920 (2007).
• 16) Wang L, Wible BA, Wan X, Ficker E. Cardiac glycosides as novel inhibitors of human ether-a-go-go-related gene channel trafficking. J. Pharmacol. Exp. Ther., 320, 525–534 (2007).
• 17) Dennis A, Wang L, Wan X, Ficker E. hERG channel trafficking: novel targets in drug-induced long QT syndrome. Biochem. Soc. Trans., 35, 1060–1063 (2007).
• 18) van der Heyden MA, Smits ME, Vos MA. Drugs and trafficking of ion channels: a new pro-arrhythmic threat on the horizon. Br. J. Pharmacol., 153, 406–409 (2008).
• 19) Ishii K, Norota I, Obara Y. Endocytic regulation of voltage-dependent potassium channels in the heart. J. Pharmacol. Sci., 120, 264–269 (2012).
• 20) Milligan CJ, Li J, Sukumar P, Majeed Y, Dallas ML, English A, Emery P, Porter KE, Smith AM, McFadzean I, Beccano-Kelly D, Bahnasi Y, Cheong A, Naylor J, Zeng F, Liu X, Gamper N, Jiang LH, Pearson HA, Peers C, Robertson B, Beech DJ. Robotic multiwell planar patch-clamp for native and primary mammalian cells. Nat. Protoc., 4, 244–255 (2009).
• 21) Classen S, Schwarzer S, Ludwig J, Zünkler BJ. Electrophysiological and fluorescence microscopy studies with HERG channel/EGFP fusion proteins. J. Membr. Biol., 222, 31–41 (2008).
• 22) Warmke J, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc. Natl. Acad. Sci. U.S.A., 91, 3438–3442 (1994).
• 23) Masumiya H, Saito M, Ito M, Matsuda T, Noguchi K, Iida-Tanaka N, Tanaka H, Shigenobu K. Lack of action potential-prolonging effect of terfenadine on rabbit myocardial tissue preparations. Biol. Pharm. Bull., 27, 131–135 (2004).
• 24) Zeng H, Penniman JR, Kinose F, Kim D, Trepakova ES, Malik MG, Dech SJ, Balasubramanian B, Salata JJ. Improved throughput of PatchXpress hERG assay using intracellular potassium fluoride. Assay Drug Dev. Technol., 6, 235–241 (2008).
• 25) Iida-Tanaka N, Namekata I, Kaneko M, Tamura M, Kawanishi T, Nakamura R, Shigenobu K, Tanaka H. Involvement of intracellular Ca²⁺ in the regulatory volume decrease after hyposmotic swelling in MDCK cells. J. Pharmacol. Sci., 104, 397–401 (2007).
• 26) Iida-Tanaka N, Namekata I, Tamura M, Kawamata Y, Kawanishi T, Tanaka H. Membrane-labeled MDCK cells and confocal microscopy for the analyses of cellular volume and morphology. Biol. Pharm. Bull., 31, 731–734 (2008).
• 27) Tsuneoka Y, Kobayashi Y, Honda Y, Namekata I, Tanaka H. Electrical activity of the mouse pulmonary vein myocardium. J. Pharmacol. Sci., 119, 287–292 (2012).
• 28) Hamaguchi S, Kawakami Y, Honda Y, Nemoto K, Sano A, Namekata I, Tanaka H. Developmental changes in excitation-contraction mechanisms of the mouse ventricular myocardium as revealed by functional and confocal imaging analyses. J. Pharmacol. Sci., 123, 167–175 (2013).