Modulation of HERG Potassium Channels by Extracellular Magnesium and Quinidine : Journal of Cardiovascular Pharmacology

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Modulation of HERG Potassium Channels by Extracellular Magnesium and Quinidine

Po, S. S.; Wang, D. W.; Yang, Iris Chun-Hui; Johnson, J. P. Jr.; Nie, Li; Bennett, P. B.

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Journal of Cardiovascular Pharmacology: February 1999 - Volume 33 - Issue 2 - p 181-185
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Abstract

A common arrhythmia associated with a prolonged QT interval observed in patients receiving certain class Ia or class III antiarrhythmic agents is known as torsades de pointes (1,2). Drug-induced prolongation of ventricular repolarization has been referred to as the acquired long QT syndrome (aLQT) to contrast it with the congenital forms. Congenital LQT represents a heterogeneous group of disorders with different molecular mechanisms (3-11). A link between the acquired and congenital LQT syndromes involves the cardiac delayed rectifying current known as IKr(5,12-14). The molecular basis of IKr was recently demonstrated to involve a K+ channel subunit encoded by the human ether-a-go-go-related gene or HERG(12,15). Because IKr plays a key role in normal cellular repolarization, suppression of this potassium current could result in aLQT. Intravenous Mg2+ is an effective acute therapy for torsades de pointes (16-23). Therefore we investigated the actions of quinidine and Mg2+ on the HERG potassium channel currents.

METHODS

HERG cDNA was provided by Dr. Mark Keating (University of Utah). The cDNA was subcloned into a modified oocyte expression vector (24) for in vitro RNA transcription and expression in Xenopus laevis oocytes. HERG cDNA was linearized with EcoRI, and cRNA was synthesized with SP6 polymerase by using a transcription kit (Cap-Scribe; Boehringer Mannheim, Indianapolis, IN, U.S.A.). Oocytes were pressure injected with 1-5 ng of in vitro transcribed cRNA in a volume of ∼40 nl. For transfection into mammalian cells, HERG cDNA was subcloned into the pBK/CMV plasmid vector (Stratagene, La Jolla, CA, U.S.A.). hKv1.5 was stably expressed in a mouse Ltk cell line (25). The bath solution (ND-96) for two-electrode voltage clamp, internal and bath (Tyrode's) solution for whole-cell patch clamp were described previously (25-27). Both ND-96 and Tyrode's solution contained 1 mM of Mg2+ unless indicated otherwise. Different concentrations of Mg2+ were made by the addition of aliquots of 1 M MgCl2 or MgSO4 stock solution. Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

The methods of two-electrode voltage clamp and whole-cell patch clamp (28) were described previously (25-27,29). In brief, cells were held at −80 mV. Channel opening was assessed by voltage-clamp steps to test membrane potentials (from +50 to −80 mV) followed by a step to −70 mV (oocytes) or −50 mV (tsA-201 cells) to assess the K+ tail currents. The tail-current amplitudes were plotted as a function of the test potentials to estimate the voltage dependence of channel opening (voltage-activation curves). These data were fit with a Boltzmann equation: Equation (1) where V and V1/2 represent the test membrane potential and membrane potential where the Boltzmann function equals 0.5 · Imax, respectively. The parameters R, T, and F have their usual thermodynamic meanings; z is the apparent effective charge valence for the relation, which determines the steepness of the curve. Comparisons were made by a paired t test. Differences between means were considered statistically significant at p < 0.05.

RESULTS

Extracellular Mg2+ decreases HERG potassium currents

At present it is not understood under what circumstances suppression of K+ current is proarrhythmic (2,30-32). Nevertheless, it is reasonable to propose that reversal of drug-induced K+ current suppression could be antiarrhythmic is this setting. Therefore our experiments were designed to investigate two distinct hypotheses for the actions of extracellular Mg2+ on the HERG potassium current. First, extracellular Mg2+ exerts its ameliorative action by a direct action on the HERG potassium channel by enhancement of potassium current. Second, the beneficial effects of Mg2+ may ensue from displacement of the drug that has suppressed the potassium current, resulting in an enhancement of potassium current (reversal of block).

We first tested the direct effects of extracellular Mg2+ on two distinct voltage-gated human cardiac potassium channels: HERG and the human Kv1.5 delayed rectifier potassium channel. hKv1.5 is thought to underlie a rapid component of delayed rectification in human heart (25,33,34). Divalent cations can have nonspecific effects to screen membrane surface charges and alter surface potential (35); therefore we compared these two channels with the hope of distinguishing nonspecific surface charge screening (which should occur in each channel) from specific HERG-Mg2+ interactions.

These experiments were completed by measuring voltage-activation curves in different extracellular Mg2+ concentrations. This gave an estimate of the membrane potential dependence for channel opening (slope factor or equivalent valence, z), the membrane potential range over which channels opened (quantified by the V1/2 parameter), and the relative maximal number of conducting potassium channels at depolarized membrane potentials. The data from each cell were normalized to the maximal current measured in 1 mM extracellular Mg2+. In the presence of 0.3 mM extracellular Mg2+, the tail current of HERG was 11.4 ± 0.5% (n = 3) greater than in 1 mM. The HERG potassium channel tail current was suppressed in 3 mM Mg2+ and 10 mM Mg2+ by 12.4 ± 1.9% (n = 3) and 34.1 ± 5.3% (n = 4), respectively (Fig. 1A), compared with that measured in 1 mM external Mg2+. Extracellular Mg2+ did not alter the voltage sensitivity (z) of channel opening; however, the midpoint (V1/2) of the relations were shifted toward more positive membrane potentials by Mg2+(Table 1). Thus Mg2+ caused the HERG potassium channels to open at more positive membrane potentials (Fig. 1A), leading to an apparent concentration-dependent suppression of the HERG potassium current.

F1-2
FIG. 1:
Effects of extracellular Mg2+ and quinidine on HERG potassium channels expressed in Xenopus laevis oocytes. A: Effects of Mg2+ on HERG currents expressed in oocytes studied by conventional two-electrode voltage clamp (Methods). Oocytes were held at −80 mV and then given a voltage step to 50 mV for 200 ms followed by return to −70 mV for 5 s. B: Effects of Mg2+ on current suppressed by quinidine. Oocytes were superfused with 3 μM quinidine in 1 mM Mg2+. After steady-state block was achieved, additional 2 mM MgSO4 was washed in (3 mM Mg2++ in total) in the continued presence of 3 μM quinidine.
T1-2
TABLE 1:
Modulation HERG channel activation by extracellular Mg2

In similar experiments with human Kv1.5 channels, we found no significant differences in the potassium currents recorded in 1 or 3 mM extracellular Mg2+. The midpoints (V1/2) of the voltage-activation curves were −9.4 ± 0.95 (n = 3) and −7.6 ± 0.95 (n = 3) in these concentrations of Mg2+, respectively. Increasing Mg2+ to 10 mM shifted the V1/2 of this relation to −2.8 ± 0.5 mV (n = 3). The magnitude of the measured currents was not altered in Mg2+ concentrations between 0.3 and 3 mM (p > 0.1, paired t test; n = 3). At the highest concentrations tested, 10 mM, hKv1.5 currents were decreased by 6% to 94 ± 3.0% of the level observed in 1 mM. This decrease was statistically significant (p < 0.05, paired t test; n = 3). Thus although Mg2+ had small effects on hKv1.5 K+ currents, they can largely be explained by Mg2+ binding to negative surface charges and altering the membrane potential sensed by the channels. In contrast, the effects of Mg2+ on HERG K+ currents appeared to be greater and more specific, as if Mg2+ directly interacts with the HERG protein and is not acting solely through nonspecific screening of diffuse negative membrane surface charges.

Inhibitions by quinidine and extracellular Mg2+ are additive

The experiments described demonstrate that extracellular Mg2+ does not act through enhancement of HERG K+ current, and therefore this cannot be the mechanism for reversal of drug-induced torsades de pointes. They did not address whether extracellular Mg2+ can reverse potassium channel block by an antiarrhythmic agent. For these experiments, we used quinidine because it is the most frequently prescribed antiarrhythmic agent, and it is associated with development of torsades de pointes (36). MgSO4 rather than MgCl2 was used because it is conceivable that sulfate rather than Mg2+ is the active component. Figure 1B demonstrates that quinidine (3 μM) inhibited the K+ current through HERG channels expressed in X. laevis oocytes by 32 ± 3.2% (n = 5). Increasing the Mg2+ concentration to 3 mM further suppressed the HERG currents by an additional 10 ± 1.1% (n = 5). To ensure that this Mg2+ effect was not related to the heterologous expression system used, HERG channels also were expressed in a cultured human cell line (tsA-201 cells). Similar qualitative results were observed: increasing concentrations of extracellular Mg2+(Fig. 2A) or quinidine each inhibited HERG currents (Fig. 2B). MgSO4 (3 mM) suppressed the HERG current by 15.1 ± 1.5% (n = 8), and 10 mM MgSO4 decreased the HERG current by 30.6 ± 2.4% (n = 7). Quinidine suppressed HERG current with a median inhibitory concentration (IC50) of 0.32 ± 0.01 μM (n = 12). Increasing extracellular Mg2+ from 1 to 3 mM inhibited HERG currents by an additional 14.5 ± 1.4% (n = 13). In one additional experiment, 10 mM MgSO4 caused an additional 36% suppression of currents. In similar experiments with hKv1.5 (Fig. 2C), significant block was induced by 10 μM quinidine (37), but little additional suppression of current occurred on increase of Mg2+ to 3 mM in the continued presence of quinidine (Fig. 2E). The IC50 for quinidine suppression of Kv1.5 current was 6.0 ± 0.4 μM (n = 5). We found no differences between MgCl2 and MgSO4, suggesting that the effects observed were due to Mg2+ and not to its anion partner.

F2-2
FIG. 2:
Effects of extracellular Mg2+ and quinidine on HERG and hKv1.5 potassium channels expressed in cultured mammalian cells. A: Mg2+ inhibition of HERG currents in tsA-201 cells examined by whole-cell patch clamp (Methods). Cells were held at −80 mV, stepped to +50 mV for 2 s. Membrane potential was then changed to −50 mV. B: Mg2+ and quinidine block are additive. Current was suppressed by 1 μM quinidine. Increasing Mg2+ from 1 to 3 mM during continued exposure to quinidine caused an additional suppression of K+ current. C: Recording of hKv1.5 (HK2) potassium current under control conditions. D: Suppression of hKv1.5 potassium current by 10 μM quinidine in the presence of 1 mM (D) or 3 mM (E) Mg2+. The holding potential was −80 mV. Families of potassium currents are shown superimposed during clamp steps between −50 and +50 mV, followed by a step to −50 mV to record deactivating tail currents.

DISCUSSION

Intravenous MgSO4 is an effective therapy for torsades de pointes (16,19-23). However, the cellular electrophysiologic basis for this effect is not known. Most reported cases using intravenous MgSO4 to treat torsades de pointes involve the acquired (drug-induced) LQT. Offending drugs include quinidine, dofetilide, procainamide, and imipramine, among others (31,38). Data regarding the use of intravenous MgSO4 in the congenital LQT are limited, but one case report suggests efficacy (39).

Action-potential prolongation through suppression of the cardiac delayed rectifier known as IKr is thought to be one mechanism of antiarrhythmic drug action; however, under certain conditions, IKr suppression also plays a critical role in acquired LQT and ventricular arrhythmias (5,12,30,32,40). Recent evidence indicates that the HERG protein is the ion channel subunit responsible for IKr(12). One working hypothesis for this study was that Mg2+ may enhance HERG potassium currents, causing reversal of the drug-induced action-potential prolongation. We found that HERG currents were not enhanced and in fact were suppressed by 3 or 10 mM extracellular Mg2+ relative to the level measured in 1 mM. Magnesium caused the voltage dependence for K+ channel opening to be shifted to more depolarized membrane potentials, leading to fewer channels being open at a given membrane potential.

Ho et al. (41) investigated the effects of Ca2+ and Mg2+ on IK in rabbit SA node cells in 140 mM extracellular potassium. This current has some characteristics similar to IKr, although there are also significant differences. HERG channel currents and native IKr are greatly affected by extracellular K+ concentrations (13,42-45); however, the SA node K+ currents are not (41). Also, the SA nodal K+ current, unlike HERG, loses deactivation gating (41) in the absence of divalent cations (Ca2+ and Mg2+). Ho et al. (41) suggested that Ca2+ and Mg2+ block the channel at a site deep in the electrical field and cause channel deactivation (41). Their findings on the SA nodal K+ currents are in contrast with the interpretation that the decrease in HERG channel currents is caused by a shift in the voltage-dependent activation gating by divalent cations. The effects of Ca2+ and Mg2+ on HERG channel currents can be explained entirely by changes in gating kinetics and not by direct block of the channel (46).

The effects of external Mg2+ on HERG potassium currents suggest that the therapeutic benefits of intravenous Mg2+ do not derive from modulation of HERG channels. Some of the observed Mg2+ effects appear specific for the HERG protein because extracellular Mg2+ had a much greater effect on HERG channels compared with another cardiac delayed rectifier, hKv1.5. Nonspecific screening of membrane-surface negative charges (e.g., membrane phospholipid head groups) by the positively charged magnesium will alter the transmembrane potential sensed by the channel protein. This will affect any ion channel in the membrane. In contrast, specific binding of Mg2+ to the HERG protein would result in the relatively greater effect that we observe. These findings warrant further investigation. Extracellular Mg2+ does not act by relieving block by quinidine. On the contrary, additional inhibition was observed in the presence of 3 or 10 mM Mg2+. This study represents the first comparison of quinidine block on two important but distinct cardiac delayed rectifier channels. We found that quinidine profoundly inhibits the HERG K+ current, much more than has been reported for IKs or the sodium channel, even though quinidine is a class Ia antiarrhythmic agent. Previous studies showed that quinidine inhibits sodium channels (47) and IKs with an IC50 of ∼50 μM(29) and Kv1.5 with an IC50 of 6 μM(29). Our results confirm these data with Kv1.5 and show that HERG channels are blocked with an IC50 that is more than an order of magnitude lower (0.3 μM). The data support the hypothesis that quinidine-induced aLQT most likely involves block of HERG channels.

These observations demonstrate that mechanisms other than HERG modulation play a role in the reversal of torsades de pointes by magnesium. Plausible mechanisms include enhancement of another cardiac potassium current by Mg2+. Alternatively, the cellular mechanism may involve suppression of an inward current such as that mediated by L-type calcium channels (48,49). Future investigation should be directed toward elucidating these mechanisms.

Acknowledgment: We thank Erin Reich for help with the Kv1.5 experiments, M. Choi and B. Palmer for technical assistance, and Dr. Dan Roden for reviewing the manuscript before submission. This work was supported by grants HL51197 and HL46681 from the National Institutes of Health and by a Grant-In-Aid from the American Heart Association. Dr. Bennett was an Established Investigator of the American Heart Association during the course of this study.

REFERENCES

1. Towbin JA, Li H, Taggart RT, et al. Evidence of genetic heterogeneity in Romano-Ward long QT syndrome: analysis of 23 families. Circulation 1994;90:2635-44.
2. Ben-David J, Zipes DP. Torsades de pointes and proarrhythmia. Lancet 1993;341:1578-82.
3. Benson DW, MacRae CA, Vesely MR, et al. Missense mutation in the pore region of HERG causes familial long QT syndrome. Circulation 1996;93:1791-5.
4. Curran M, Atkinson D, Timothy K, et al. Locus heterogeneity of autosomal dominant long QT syndrome. J Clin Invest 1993;92:799-803.
5. Sanguinetti MC, Curran ME, Spector PS, Keating MT. Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia [published erratum appears in Proc Natl Acad Sci U S A 1996;93:8796]. Proc Natl Acad Sci U S A 1996;93:2208-12.
6. Schwartz PJ, Priori SG, Locati EH, et al. Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na+ channel blockade and to increases in heart rate: implications for gene-specific therapy [Comments]. Circulation 1995;92:3381-6.
7. Wang Q, Shen J, Splawski I, et al. SCN5A mutations associated with an inherited cardiac arrhythmia, long QT syndrome. Cell 1995;80:805-11.
8. Bennett PB, Yazawa K, Makita N, George AL Jr. Molecular mechanism for an inherited cardiac arrhythmia [Comments]. Nature 1995;376:683-5.
9. Spector PS, Curran ME, Keating MT, Sanguinetti MC. Class III anti-arrhythmic drugs block HERG: a human cardiac delayed rectifier K+ channel: open-channel block by methanesulfonanilides. Circ Res 1996;78:499-503.
10. Wang Q, Curran ME, Splawski I, et al. Positional cloning of a novel potassium channel gene: KVLQT1 mutations cause cardiac arrhythmias. Nature Genet 1996;12:17-23.
11. Wang Q, Shen J, Li Z, et al. Cardiac sodium channel mutations in patients with long QT syndrome, an inherited cardiac arrhythmia. Hum Mol Genet 1995;4:1603-7.
12. Sanguinetti MC, Jiang C, Curran ME, Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 1995;81:299-307.
13. Sanguinetti MC, Jurkiewiez NK. Delayed rectifier outward K+ current is composed of two currents in guinea pig atrial cells. Am J Physiol 1991;260:H393-9.
14. Sanguinetti MC, Jurkiewiez NK. Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 1990;96:195-215.
15. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A 1994;91:3438-42.
16. Banai S, Schuger C, Benhorin J, Tzivoni D. Treatment of torsades de pointes with intravenous magnesium [Letter]. Am J Cardiol 1989;63:1539-40.
17. Moss AJ. Molecular genetics and ventricular arrhythmias [Editorial; comment]. N Engl J Med 1992;327:885-7.
18. Moss AJ, Robinson JL. Clinical aspects of the idiopathic long QT syndrome. Ann N Y Acad Sci 1992;644:103-11.
19. Tzivoni D, Keren A, Cohen AM, et al. Magnesium therapy for torsades de pointes. Am J Cardiol 1984;53:528-30.
20. Keren A, Tzivoni D. Magnesium therapy in ventricular arrhythmias. Pacing Clin Electrophysiol 1990;13:937-45.
21. Moss AJ, Schwartz PJ, Crampton RS, et al. The long QT syndrome: prospective longitudinal study of 328 families. Circulation 1991;84:1136-44.
22. Stern S, Keren A, Tzivoni D. Torsades de pointes: definitions, causative factors, and therapy: experience with sixteen patients. Ann N Y Acad Sci 1984;427:234-40.
23. Tzivoni D, Banai S, Schuger C: Treatment of torsades de pointes with magnesium sulfate. Circulation 1988;77:392-7.
24. Krieg PA, Melton DA. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol 1987;155:397-415.
25. Snyders DJ, Tamkun MM, Bennett PB. A rapidly activating and slowly inactivating potassium channel cloned from human heart: functional analysis after stable mammalian cell culture expression. J Gen Physiol 1993;101:513-43.
26. Po S, Roberds S, Snyders DJ, Tamkun MM, Bennett PB. Heteromultimeric assembly of human potassium channels: molecular basis of a transient outward current? Circ Res 1993;72:1326-36.
27. Po S, Snyders DJ, Baker R, Tamkun MM, Bennett PB. Functional expression of an inactivating potassium channel cloned from human heart. Circ Res 1992;71:732-6.
28. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 1981;391:85-100.
29. Balser JR, Bennett PB, Hondeghem LM, Roden DM. Suppression of time-dependent outward current in guinea pig ventricular myocytes: actions of quinidine and amiodarone. Circ Res 1991;69:519-29.
30. Rosen MR, Schwartz PJ, for Task Force of the Working Group on Arrhythmias of the European Society of Cardiology. The Sicilian gambit: a new approach to the classification of antiarrhythmic drugs based on their actions on arrhythmogenic mechanism. Circulation 1991;84:1831-51.
31. Lazzara R. Antiarrhythmic drugs and torsades de pointes. Eur Heart J 1993;14(suppl H):88-92.
32. Roden DM, Lazzara R, Rosen M, Schwartz PJ, Towbin J, Vincent GM. Multiple mechanisms in the long-QT syndrome: current knowledge, gaps, and future directions: the SADS Foundation task force on LQTS. Circulation 1996;94:1996-2012.
33. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes: evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circ Res 1993;73:1061-76.
34. Bennett PB, Po S, Snyders DJ, Tamkun MM. Molecular and functional diversity of cloned cardiac potassium channels. Cardiovasc Drugs Ther 1993;7(suppl 3):585-92.
35. Hille B, Woodhull AM, Shapiro BI. Negative surface charge near the sodium channels of nerve: divalent ions, monovalent ions, and pH. Trans R Soc Lond 1975;270:301-18.
36. Roden DM. Risks and benefits of antiarrhythmic therapy. [Review]. N Engl J Med 1994;331:785-91.
37. Snyders DJ, Knoth KM, Roberds SL, Tamkun MM. Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel. Mol Pharmacol 1992;41:322-30.
38. Roden DM. Clinical features of arrhythmia aggravation by antiarrhythmic drugs and their implications for basic mechanisms. Drug Dev Res 1990;19:153-72.
39. Garcia-Rubira JC, Lopez Garcia-Aranda V, Cruz Fernandez JM. Magnesium sulphate for torsades de pointes in a patient with congenital long QT syndrome. Int J Cardiol 1990;27:282-3.
40. Roy M, Dumaine R, Brown AM. HERG, a primary human ventricular target of the nonsedating antihistamine terfenadine [Comments]. Circulation 1996;94:817-23.
41. Ho WK, Earm YE, Lee SH, Brown HF, Noble D. Voltage- and time-dependent block of delayed rectifier K+ current in rabbit sinoatrial node cells by external Ca2+ and Mg2+. J Physiol 1996;494:727-42.
42. Smith PL, Baukrowitz T, Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel [Comments]. Nature 1996;379:833-6.
43. Wang S, Morales MJ, Liu S, Strauss HC, Rasmusson RL. Modulation of HERG affinity for E-4031 by [K+]o and C-type inactivation. FEBS Lett 1997;417:43-7.
44. Wang S, Liu S, Morales MJ, Strauss HC, Rasmusson RL. A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes. J Physiol 1997;502:45-60.
45. Schonherr R, Heinemann SH. Molecular determinants for activation and inactivation of HERG, a human inward rectifier potassium channel. J Physiol 1996;493:635-42.
46. Johnson JP, Bennett PB. Modulation of the HERG K+ channel by external divalent cations. Biophys J 1998;74:A212.
47. Snyders DJ, Hondeghem LM. Effects of quinidine on the sodium current of guinea pig ventricular myocytes: evidence for a drug-associated rested state with altered kinetics. Circ Res 1990;66:565-79.
48. Kuo CC, Hess P. Block of the L-type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells. J Physiol 1993;466:683-706.
49. Lansman JB, Hess P, Tsien RW. Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+: voltage and concentration dependence of calcium entry into the pore. J Gen Physiol 1986;88:321-47.
Keywords:

Antiarrhythmic drug; Potassium channel; Arrhythmia; Human ether-a-go-go-related gene; Long QT syndrome; LQT2; Mg2+; Torsades de pointes

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