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Inhibition of HERG channels by the local anaesthetic articaine

Siebrands, C. C.*†; Friederich, P.*

European Journal of Anaesthesiology: February 2007 - Volume 24 - Issue 2 - p 148–153
doi: 10.1017/S0265021506001311
Original Article

Background and objective: Articaine is an amide local anaesthetic widely used in dentistry. Human ether-a-go-go-related gene (HERG) potassium channels constitute potential targets involved in cardiotoxic side-effects of various pharmacological agents including amide local anaesthetics. The aim of this study was to determine the sensitivity of HERG channels to the inhibitory action of articaine and to further evaluate the effect of the mutations Y652A and F656A in the putative drug-binding region of HERG on the sensitivity for articaine.

Methods: We examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by articaine. Whole cell patch-clamp recordings were performed at room temperature.

Results: Inhibition of HERG wild-type and HERG Y652A channels by articaine was concentration dependent and reversible. The concentration-response data were described by Hill functions (wild type: IC50 = 224 ± 6 μmol L−1, Hill coefficient h = 1.17 ± 0.03, n = 23; Y652A: IC50 = 360 ± 48 μmol L−1, h = 0.93 ± 0.08, n = 26). The mutation Y5652A decreased the sensitivity by factor 1.6. The mutation F656A decreased inhibition of inward tail currents by 300 μmol L−1 articaine in 100 mmol extracellular K+ 3-fold.

Conclusions: Our results indicate that the local anaesthetic articaine does not inhibit HERG channels at clinically relevant concentrations. Articaine may therefore constitute a safer alternative for local and regional anaesthesia. The aromatic amino acid F656 rather than Y652 in the S6 region might play a role in interaction of the drug with the channel.

*University Medical Center Hamburg-Eppendorf, Department of Anaesthesiology, Hamburg, Germany

University Medical Center Hamburg-Eppendorf, Institute for Neural Signal Transduction, Hamburg, Germany

Correspondence to: Patrick Friederich, Department of Anaesthesiology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20251 Hamburg, Germany. E-mail:; Tel: +49 40 42803 6414; Fax: +49 40 42803 5024

Accepted for publication 31 July 2006

First published online 29 August 2006

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Articaine belongs to the class of amide local anaesthetics. However, in contrast to bupivacaine and ropivacaine articaine contains a thiophene ring instead of the benzene ring and an additional ester group. Articaine is rapidly hydrolysed to the ineffective metabolite articainic acid by plasma esterases [1]. Articaine is widely used in dentistry [2], eye surgery [3–6], for tumescent local anaesthesia [7] as well as for intravenous (i.v.) regional anaesthesia [8].

Human ether-a-go-go-related gene (HERG) codes for the pore forming component of the rapid delayed rectifier current (IKr) in the heart [9]. IKr plays an important role in the repolarization of cardiac action potentials and mutations in the HERG gene may lead to prolongation of the action potential resulting in the congenital long QT syndrome [9]. HERG channels constitute toxicologically relevant targets for many structurally and functionally unrelated substances such as antiarrhythmic drugs [10,11], antihistamines [12,13], psychoactive drugs [14], gastrointestinal prokinetic agents [15,16], macrolide antibiotics [17] and local anaesthetics [18–21]. Pharmacological inhibition of IKr may cause drug induced long QT syndrome, severe ventricular dysrhythmia and sudden cardiac arrest [22].

The structural reason for the inhibition of HERG channels by many structurally different pharmacological agents is the large pore forming cavity of HERG channels, allowing preferential trapping of many structurally unrelated drugs [22]. The aromatic amino acids tyrosine 652 and phenylalanine 656 in the S6 transmembrane domain of the channel were identified to mediate high-affinity drug binding to HERG [22] presumably by hydrophobic interaction with Phe656 and π-cation interaction or π-stacking with Tyr652 [23]. In a previous study we were able to demonstrate that these aromatic amino acids also play a role in interaction of amino-amide local anaesthetics with HERG channels [21].

In contrast to other amide local anaesthetics and despite the widespread use of articaine in clinical medicine, the effects of articaine on human cardiac ion channels have not been evaluated. The aim of this study therefore was to investigate the sensitivity of HERG potassium channels to the inhibitory action of the local anaesthetic articaine. It was furthermore intended to establish if and to what extent the aromatic amino acids Tyr652 and Phe656 in the S6 region influence drug affinity.

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Chinese hamster ovary (CHO) cells were cultured in 50 mL flasks (NUNC; Roskilde, Denmark) at 37°C in MEM Alpha medium (GIBCO; Invitrogen, Carlsbad, CA, USA) with 10% fetal calf serum, penicillin 100 IU mL−1 and streptomycin 100 mg mL−1 in a humidified atmosphere (5% CO2). Cells were sub-cultured in 35 mm diameter monodishes (NUNC; Roskilde, Denmark) at least 1 day before transfection.

The mutants HERGY652A and HERGF656A were created by site-directed mutagenesis. All channels were cloned in the pcDNA3 expression vector. CHO cells were transiently transfected with 1 μg HERG wild-type or mutant cDNA, 0.5 μg EFGP cDNA and 3 μL lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) per dish according to the manufacturer's protocol after 1 day. Cells were co-transfected with an EGFP pcDNA3 construct to verify successful transfection. Only green fluorescing cells were used for patch-clamp experiments. Patch-clamp experiments were performed 1 or 2 days after transfection.

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Whole-cell currents were recorded using the patch-clamp technique with an EPC-9 amplifier and Pulse software version 8.50 (HEKA Elektronik, Lambrecht, Germany). Patch electrodes were pulled from borosilicate glass capillary tube (World Precision Instruments, Saratoga, FL, USA) on a horizontal puller (P-97; Sutter Instrument Co., Novato, CA, USA) and had a pipette resistance of 1.5–3.5 MΩ. The internal solution contained 160 mmol L−1 KCl, 0.5 mmol L−1 MgCl2, 10 mmol L−1 HEPES and 2 mmol L−1 Na-ATP (all from Sigma, Deissenhofen, Germany), adjusted to pH 7.2 with KOH. The external solution contained 135 mmol L−1 NaCl, 5 mmol L−1 KCl, 2 mmol L−1 CaCl2, 2 mmol L−1 MgCl2, 5 mmol L−1 HEPES, 10 mmol L−1 sucrose and 0.1 mg mL−1 phenol red (all from Sigma, Deissenhofen, Germany) adjusted to pH 7.4 with NaOH. In order to record inward tail currents of HERG channels, an extracellular solution with high [K+] was used, containing 40 mmol L−1 NaCl, 100 mmol L−1 KCl, 2 mmol L−1 CaCl2, 2 mmol L−1 MgCl2, 5 mmol L−1 HEPES, 10 mmol L−1 sucrose and 0.1 mg mL−1 phenol red, adjusted to pH 7.4 with NaOH.

Series resistance was 2.5–6.0 MΩ and was actively compensated for by 85%. A leak subtraction protocol was used except for recordings with high extracellular [K+]. The recorded signal was filtered at 2 kHz and stored with a sampling rate of 5 kHz for analysis. Articaine (Aventis, Frankfurt, Germany) was dissolved in the extracellular solution. A hydrostatically driven perfusion system was used to apply the drug onto the cells and to exchange the extracellular solutions. All experiments were performed at room temperature.

Different pulse protocols were used to establish the pharmacological sensitivities of the channels. The holding potential was −80 mV for all experiments. For the pharmacological experiments with HERGwt and HERGY652A, a ramp protocol was used [24]. Cells were depolarized from a holding potential of −80 to +60 mV and repolarized to −80 mV within 1 s. In high extracellular [K+], a single pulse to +20 mV for 2 s and a tail potential of −120 mV for 1 s was used for the pharmacological tests. Repetitive pulses were applied to determine that steady-state inhibition was reached.

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Data analysis

Data were analysed with Pulse Fit software (HEKA Elektronik, Lambrecht, Germany) and with Kaleidagraph software (Synergy Software, Reading, PA, USA). The inhibition of currents was quantified by the reduction of the maximal current during the ramp protocol or during the tail pulse in case of the high extracellular [K+] experiments. In addition, the reduction of charge flow during the ramp protocol was analysed. The charge crossing the membrane is equivalent to the time integrals of current traces and was determined using Pulse Fit software. The fractional block f was calculated by the following formula: f = 1 − [2 × Imax, drug/(Imax, control + Imax, wash out)]. Concentration–response curves were fitted by a Hill function: f = 1/[1 + (IC50/c)h], where IC50 is the concentration of half-maximal inhibition, c is the concentration of the local anaesthetic and h is the Hill coefficient. Statistical significance was tested using a two-sided Student's t-test (Excel; Microsoft, Redmond, WA, USA). Data are presented as mean ± SD unless stated otherwise; n values indicate the number of experiments.

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The inhibitory effect of the local anaesthetic articaine on HERGwt and HERGY652A currents was investigated using a ramp protocol [18,21,24]. This protocol evoked bell shaped currents (Fig. 1a). The mean time to peak current was 717 ± 26 ms for HERGwt (n = 21) and 600 ± 24 ms for HERGY652A (n = 24). These values correspond to peak voltages of −40 ± 4 mV (HERGwt) and of −24 ± 3 mV (HERGY652A), respectively. Articaine reduced HERGwt and HERGY652A currents in a concentration-dependent and reversible manner (Fig. 1a,b). The inhibition was quantified as the decrease of the maximum current as well as the reduction of the charge flow during the depolarization. The concentration-response data were mathematically described by Hill functions (Fig. 1b). Parameters of the Hill functions are presented in Table 1. The mutation HERGY652A increased the concentration of half-maximal inhibition (IC50) by a factor of 1.6 for inhibition of maximum current and by 1.8 for inhibition of charge flow compared to the wild-type channel. The Hill coefficients were close to unity for inhibition of both channels. Application of articaine caused a concentration-dependent and reversible rightward shift of the peak HERGwt current and therefore increased the time to peak current response. In contrast, the application of articaine caused a concentration-dependent and reversible leftward shift of the peak HERGY652A current and therefore decreased the time to peak current response. For example, 1 mmol L−1 articaine caused a shift of the time to peak HERGwt current response of +84 ± 19 ms (n = 5) and caused a shift of −90 ± 18 ms (n = 6), for HERGY652A, corresponding to a shift in voltage of +12 ± 3 mV and −13 ± 3 mV respectively.

Figure 1.

Figure 1.

Table 1

Table 1

HERGF656A channels only conducted very small currents under low extracellular [K+]. Inward tail currents at −120 mV were therefore recorded using high extracellular [K+] [21]. In order to compare all three channels, HERGwt and HERGY652A currents were recorded under these conditions as well (Fig. 2a). The effect of 300 μmol L−1 articaine which is close to the IC50-value for HERGwt was compared on HERGwt, HERGY652A and on HERGF656A (Fig. 2b). Articaine (300 μmol L−1) inhibited HERGwt by 31 ± 3%. The effect on HERGY652A was not significantly different (33 ± 5%; P > 0.05). In contrast, inhibition of the mutant HERGF656A was significantly smaller (10 ± 2%; P < 0.001) than inhibition of both HERGwt and HERGY652A (Fig. 2b).

Figure 2.

Figure 2.

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In this study we established the effects of the local anaesthetic articaine on the human cardiac potassium channel HERG. HERG channels constitute potential molecular targets for the cardiotoxic action of various drugs [22] including local anaesthetics [18–21]. Like other amino-amide local anaesthetics such as bupivacaine, ropivacaine and mepivacaine [18–21], articaine inhibited HERG channels in a concentration-dependent and reversible manner. High extracellular potassium reduced the inhibition of HERGwt and HERGY652A by articaine. This effect has been described before for the high-affinity blocker E-4031 and might indicate that articaine acts as an open pore blocker [25].

HERG channels were 10-fold less sensitive to articaine than to bupivacaine. This difference in inhibitory potency between bupivacaine and articaine might result from differences in molecular structure as well as in lipophilicity, since it has been demonstrated that the lipophilicity of amino-amide local anaesthetics correlates well with their potency to block HERG channels [21]. We analysed the correlation between the lipophilicity of the local anaesthetics represented as the octanol: buffer coefficient (log P) [2,26] and the log IC50 using the values for a homologue series of local anaesthetics established previously [21]. This correlation was extended by the values for articaine obtained in this work (Fig. 3), while the linear fit only includes bupivacaine, ropivacaine and mepivacaine. For HERGwt the value for articaine fits very well with this correlation indicating that the lipophilicity is an important factor of articaine interaction with HERGwt channels. The smaller lipophilicity of articaine thus explains the lower sensitivity of HERG channels to articaine compared with bupivacaine.

Figure 3.

Figure 3.

Amino-amide local anaesthetics like bupivacaine and ropivacaine have been shown to interact with HERG channels via the aromatic amino acids Tyr652 and Phe656 in the S6 region of HERG [21]. Mutating these amino acids to alanine reduced the IC50 3–10-fold. The effect of the mutation Y652A on the sensitivity of HERG to articaine was less pronounced, the IC50 for articaine was only reduced by a factor of 1.6. Furthermore, for HERGY652A the correlation for bupivacaine, ropivacaine and mepivacaine does not meet the value for articaine very well (Fig. 3) suggesting that the interaction site of articaine on HERG channels is different from the interaction site of bupivacaine, ropivacaine and mepivacaine. Tyr652 thus only plays a minor role in hydrophobic interaction of articaine with HERG channels. A possible reason might be the different molecular structure of articaine compared to bupivacaine, ropivacaine and mepivacaine. It has been reported before for the HERG channel blocker vesnarinone that mutating Tyr652 to alanine only marginally reduced inhibition compared to HERGwt [27]. This may lead to the assumption that interaction of drugs with HERG channels is not necessarily mediated by the aromatic residue Tyr652.The mutation F656A reduced the inhibition by 300 μmol L−1 articaine by a factor of 3. Similar effects were observed for bupivacaine [21], indicating that the residue F656 is involved in interaction of articaine with HERG channels.

Inhibition of HERG channels by local anaesthetics may lead to drug induced long QT syndrome and sudden cardiac arrest in case of accidental intravascular injection [28,29]. Peak plasma concentrations of articaine in peripheral blood of 1.85 μg mL−1 (6.5 μmol L−1) were observed after i.v. regional anaesthesia [8] and of 0.26 μg mL−1 (0.9 μmol L−1) after tumescent local anaesthesia [7]. Since about 70% of articaine are bound to plasma proteins [2], the free plasma concentration will be approximately 0.1–0.5 μg mL−1 corresponding to 0.8–2.0 μmol L−1. These concentrations are far below inhibitory concentrations of HERG channels. It has been proposed that in vitro inhibition of HERG channels can serve as an indicator for potency to induce QT prolongation and torsades de pointes [30–32]. Given the fast metabolism of articaine, it seems furthermore unlikely that toxicologically relevant concentrations will easily be reached in the heart. Although articaine may at extremely high concentrations induce long-QT syndrome (LQTS) and ventricular dysrhythmia, it is tempting to speculate that this seems unlikely to be of clinical significance.

Taken together our results demonstrate that articaine blocks HERG channels with a low potency. Interaction is mediated by the aromatic residue Phe656 rather than Tyr652. Inhibitory concentrations for HERG channels are much higher than plasma concentrations of articaine occurring during clinical application [7,33]. Articaine may therefore constitute a local anaesthetic with a high margin of safety.

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This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (FR 1625/1-1), the Department of Anaesthesiology, University Medical Center Hamburg-Eppendorf, Germany, and the Institute for Neural Signal Transduction, University Medical Center Hamburg-Eppendorf, Germany. Articaine was a kind gift from Aventis, Frankfurt, Germany. The authors are grateful to Prof. Olaf Pongs, Ph.D. (Director of the Institute of Neural Signal Transduction, University Hospital Hamburg-Eppendorf, Germany) for his generous support. We thank Andrea Zaisser (Technician, Institute for Neural Signal Transduction, University Hospital Hamburg-Eppendorf, Germany) for technical assistance.

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