Anesthesiology 2005; 103:102–12 © 2005 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Local Anesthetic Interaction with Human Ether-a-go-go–related Gene (HERG) Channels Role of Aromatic Amino Acids Y652 and F656 Cornelia C. Siebrands, M.Sc.,* Nicole Schmitt, Ph.D.,† Patrick Friederich, M.D.‡ Background: Human ether-a-go-go–related gene (HERG) po- and local anesthetics.10–12 Pharmacologic inhibition of tassium channels constitute a potential target involved in car- I may cause drug-induced long QT syndrome, severe diotoxic side effects of amino-amide local anesthetics. The mo- Kr lecular interaction site of these low-affinity blockers with HERG ventricular arrhythmia, and sudden cardiac death. Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/103/1/102/358525/0000542-200507000-00017.pdf by guest on 24 September 2021 channels is currently unknown. The aim of this study was to HERG channels lack the Pro-Val-Pro motif conserved in determine the effect of the mutations Y652A and F656A in the other Kv channels that is supposed to produce a kink in putative drug binding region of HERG on the inhibition by the S6 helix.13 This results in a larger pore-forming cavity bupivacaine, ropivacaine, and mepivacaine. of HERG channels, allowing preferential trapping of Methods: The authors examined the inhibition of wild-type 14 and mutant HERG channels, transiently expressed in Chinese many structurally unrelated drugs. A scanning analysis hamster ovary cells by bupivacaine, ropivacaine, and mepiva- of the S6 transmembrane domain of the channel identi- caine. Whole cell patch clamp recordings were performed at fied two aromatic amino acids that are important for room temperature. high-affinity drug binding to HERG: tyrosine 652 and Results: Inhibition of HERG wild-type and mutant channels phenylalanine 656.2 Mutating these residues to alanine by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the increases the IC50 value of MK-499, terfenadine, and 2 order bupivacaine > ropivacaine > mepivacaine for wild-type cisapride between 100- and 650-fold. All high-affinity and mutant channels. The mutant channels were approxi- blockers (IC50 values within the nanomolar range) tested mately 4–30 times less sensitive to the inhibitory action of the are suggested to bind to this region of the channel2,15 by different local anesthetics than the wild-type channel. The con- ␲ centration–response data were described by Hill functions (bu- hydrophobic interaction with Phe656 and -cation inter- 16 .action or ␲-stacking with Tyr652 ؍ ؍ ␮ ؎ ؍ pivacaine: wild-type IC50 22 2 M,n 38; Y652A IC50 ؍ ␮ ؎ 95 5 M,n 31). The mutations resulted in a change of the The situation is different for low-affinity blockers (IC50 stereoselectivity of HERG channel block by ropivacaine. The values within the micromolar range) of HERG channels. potency of the local anesthetics to inhibit wild-type and mutant Mutating either of the aromatic amino acids to alanine channels correlated with the lipophilicity of the drug (r > 0.9). Conclusions: These results indicate that local anesthetics has severe effects on the inhibition by some compounds, 17–19 specifically but not exclusively interact with the aromatic resi- such as vesnarinone and quinidine, but not by oth- dues Y652 and F656 in S6 of HERG channels. ers, such as the selective serotonin reuptake inhibitor fluvoxamine20 and the antiparkinsonian drug budip- HUMAN ether-a-go-go–related gene (HERG) codes for ine.21 Different structural requirements have therefore the pore-forming component of the rapid delayed recti- been suggested to mediate low-affinity block of HERG 1 22 fier channel (IKr) in the heart. HERG channels constitute channels. toxicologically relevant targets for many structurally and Based on their IC50 values, amino-amide local anesthet- functionally unrelated substances, such as antiarrhyth- ics have to be regarded as low-affinity blockers of HERG mic drugs,2,3 antihistamines,4,5 psychoactive drugs,6 gas- channels10–12 with a so far unknown molecular site of trointestinal prokinetic agents,7,8 macrolide antibiotics,9 interaction. These local anesthetics differ by the length of their N-substituent, which is a butyl group (bupiva- caine), a propyl group (ropivacaine), or a methyl group * Ph.D. Student, Department of Anesthesiology and Institute for Neural Signal (mepivacaine). The length of the substituent determines Transduction, ‡ Privatdozent, Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. † Postdoctoral Researcher, the lipophilicity and may thus be a structural require- Department of Medical Physiology, The Panum Institute, University of Copenha- ment for hydrophobic interactions between the drug gen, Copenhagen, Denmark. and the channel protein. It was shown before that the Received from the Department of Anesthesiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Submitted for publication September length of the N-substituent influences the potency of 2, 2004. Accepted for publication April 20, 2005. Supported by grant No. FR local anesthetics to block Kv1.5 channels23 and HERG 1625/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany; the De- 11 partment of Anesthesiology, University Hospital Hamburg-Eppendorf, Hamburg, channels. If amino-amide local anesthetics were to Germany; and the Institute for Neural Signal Transduction, University Hospital interact with the aromatic amino acids in the S6 region, Hamburg-Eppendorf, Hamburg, Germany. Levobupivacaine, ropivacaine, and mepivacaine were gifts from AstraZeneca, So¨dertalje, Sweden. Dr. Friederich has the potency to inhibit HERG channels would be ex- received lecture/travel fees from Abbott, Wiesbaden, Germany. Presented in part pected to correlate with the lipophilic properties of the at the Annual Meeting of the German Society of Physiology, Leipzig, Germany, March 14–17, 2004. drugs. Point mutations of these aromatic amino acids Address reprint requests to Dr. Friederich: Zentrum fu¨r Ana¨sthesiologie, Uni- may furthermore be expected to alter the relation be- versita¨tsklinik Eppendorf, Martinistrasse 52, 20251 Hamburg, Germany. Address electronic mail to: [email protected]. Individual article re- tween inhibitory potency and lipophilic drug properties. prints may be purchased through the Journal Web site, www.anesthesiology.org. Inhibition of HERG channels by amino-amide local anes- Anesthesiology, V 103, No 1, Jul 2005 102 LOCAL ANESTHETIC INTERACTION WITH HERG 103 11,12 thetics is also stereoselective. If local anesthetics HEPES, 10 mM sucrose, and 0.1 mg/ml phenol red (all were to interact with the aromatic amino acids Y652 and from Sigma), adjusted to a pH of 7.4 with NaOH. To F656, mutating these amino acids might also alter stereo- record inward tail currents of HERG channels, an extra- selectivity of local anesthetic inhibition. cellular solution with high [Kϩ] was used, containing 40 Therefore, the aim of this study was to clarify whether mM NaCl, 100 mM KCl, 2 mM CaCl2,2mM MgCl2,5mM inhibition of HERG wild-type (wt) and mutant channels HEPES, 10 mM sucrose, and 0.1 mg/ml phenol red, ad- is related to the lipophilic properties of bupivacaine, justed to a pH of 7.4 with NaOH. ropivacaine, and mepivacaine. It was furthermore in- Series resistance was 2.5–6.0 M⍀ and was actively tended to establish whether and to what extent the compensated for by 85%. A leak subtraction protocol aromatic amino acids Y652 and F656 in the S6 region was used except for recordings with high extracellular ϩ ϩ influence drug affinity and stereoselective local anes- [K ] ([K ]o). The recorded signal was filtered at 2 kHz Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/103/1/102/358525/0000542-200507000-00017.pdf by guest on 24 September 2021 thetic inhibition. As a prerequisite to these experiments, and stored with a sampling rate of 5 kHz for analysis. the gating changes induced by mutating these residues2 Bupivacaine (Sigma), levobupivacaine, S(Ϫ)-ropivacaine, needed to be characterized first. The results of this study R(ϩ)-ropivacaine, and mepivacaine (all from AstraZen- may help to identify structural requirements for low- eca, So¨dertalje, Sweden) were dissolved in the extracel- affinity block of HERG channels by amino-amide local lular solution. A hydrostatically driven perfusion system anesthetics. was used to apply the drugs onto the cells and to ex- change the extracellular solutions. All experiments were performed at room temperature. Materials and Methods Different pulse protocols were used for characteriza- tion of the channels and to establish their pharmacologic Cell Culture sensitivities. The holding potential was Ϫ80 mV for all Chinese hamster ovary cells were cultured in 50 ml- experiments. For the activation protocol, cells were de- flasks (NUNC, Roskilde, Denmark) at 37°C in MEM Alpha polarized from Ϫ80 to ϩ60 mV in 10-mV steps for 1 s, medium (GIBCO; Invitrogen, Carlsbad, CA) with 10% and tail currents were recorded at Ϫ40 mV. In high fetal calf serum, 100 U/ml penicillin, and 100 mg/ml ϩ [K ] , the tail potential was changed to Ϫ120 mV. To streptomycin in a humidified atmosphere (5% CO ). o 2 analyze the deactivation, channels were activated by a Cells were subcultured in 35-mm diameter monodishes 2-s pulse to ϩ60 mV, and deactivating tail currents were (NUNC) at least 1 day before transfection. recorded at potentials from Ϫ120 to ϩ40 mV