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Block of hERG Channels by Berberine: Mechanisms of Voltage- and State-Dependence Probed With Site-Directed Mutant Channels

Rodriguez-Menchaca, Aldo MSc*; Ferrer-Villada, Tania PhD*; Lara, Jesus MD, PhD*; Fernandez, David PhD; Navarro-Polanco, Ricardo A PhD*; Sanchez-Chapula, Jose A MD, PhD*

Journal of Cardiovascular Pharmacology: January 2006 - Volume 47 - Issue 1 - p 21-29
doi: 10.1097/01.fjc.0000191564.52242.00
Original Article

Berberine prolongs the duration of cardiac action potentials without affecting resting membrane potential or action potential amplitude. Controversy exists regarding whether berberine exerts this action by preferential block of different components of the delayed rectifying potassium current, IKr and IKs. Here we have studied the effects of berberine on hERG (IKr) and KCNQ1/KCNE1 (IKs) channels expressed in HEK-293 cells and Xenopus oocytes. In HEK-293 cells, the IC50 for berberine was 3.1 ± 0.5 μM on hERG compared with 11 ± 4% decreases on KCNQ1/KCNE1 channels by 100 μM berberine. Likewise in oocytes, hERG channels were more sensitive to block by berberine (IC50 = 80 ± 5 μM) compared with KCNQ1/KCNE1 channels (∼20% block at 300 μM). hERG block was markedly increased by membrane depolarization. Mutation to Ala of Y652 or F656 located on the S6 domain, or V625 located at the base of the pore helix of hERG decreased sensitivity to block by berberine. An inactivation-deficient mutant hERG channel (G628C/S631C) was also blocked by berberine. Together these findings indicate that berberine preferentially blocks the open state of hERG channels by interacting with specific residues that were previously reported to be important for binding of more potent antagonists.

From the *Unidad “Carlos Mendez” del Centro Universitario de Investigaciones Biomédicas de la Universidad de Colima, Colima, Mexico; and †Department of Physiology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT.

Received for publication August 8, 2005; accepted October 4, 2005.

This work was supported by SEP-SESIC (Mexico) Grant No. 2003-06-001-007 and Fondo Ramon Alvarez-Buylla (Universidad de Colima, Mexico).

Reprints: Jose A. Sanchez-Chapula, CUIB, Universidad de Colima, Apdo. Postal 199, CP 28000, Colima, Col. México (e-mail: sancheza@cgic.ucol.mx).

Berberine is a benzodioxoquinolozine plant alkaloid isolated from several species of Berberis and Coptis. Various pharmacological actions have been described for berberine, including antimicrobial and cardiovascular effects. Both clinical and animal studies suggested that berberine prevented ischemia-induced ventricular tachyarrhythmia, enhanced the force of cardiac contraction, and decreased peripheral resistance and blood pressure.1 Berberine prolongs action potential duration (APD) of cardiac muscle in several species, without affecting resting membrane potential or action potential amplitude.2 In cat ventricular myocytes, the increase in APD was due to blockade of the rapid delayed rectifying potassium current, IKr.3 However, it was reported that in guinea-pig ventricular myocytes, berberine blocked the slow delayed rectifying outward potassium current, IKs without affecting IKr.4

Human ether-a-go-go related gene (hERG) subunits coassemble to form channels that conduct IKr.5 KCNQ1 and KCNE1 (KvLQT1 + minK) subunits combine to form channels that conduct IKs.6,7 We heterologously expressed these channels in HEK-293 cells and Xenopus oocytes to resolve the apparent controversy regarding the type of cardiac delayed rectifier K+ channels affected by berberine. We also determined if berberine preferentially blocked open or inactivated hERG channels because inactivation may induce conformational changes within the inner cavity that maximize interaction with some drugs.8 Finally, we determined if block was affected by mutation of specific residues (ie, Y652, F656) that are believed to be important for binding of many other drugs to hERG.9

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METHODS

Xenopus Oocytes

Wild type hERG, KCNQ1, and KCNE1 cRNAs were prepared in the pSP64 plasmid vector (Promega, Madison, WI). Point mutations were introduced into hERG (V625A, Y652A, F656T) in the pSP64 plasmid expression vector (Promega, Madison, WI) as described previously.9 G628C/S631C hERG was a gift from Dr. Gea-Ny Tseng. Complementary RNAs for injection into oocytes were prepared with SP6 Cap-Scribe (Roche, Mexico, Mexico) following linearization of the expression construct with EcoRI. Isolation and maintenance of Xenopus oocytes, and cRNA injection were performed as described.10

A GeneClamp 500 amplifier (Molecular Devices Corp) and standard 2 microelectrode voltage-clamp techniques11 were used to record currents in oocytes. Currents were recorded at room temperature (22°C -24°C), 2 to 4 days after cRNA injection. Glass microelectrodes were filled with 3 M KCl, and their tips broken to obtain resistances of 0.5 to 1 MΩ. The external low Cl- solution contained: 96 mM Mes-Na (2-[morpholino]ethanesulfonic acid salt sodium), 2 mM Mes-K, 2 mM Mes2-Ca, 5 mM, HEPES, 1 mM MgCl2, adjusted to pH 7.6 with methanesulfonic acid. Voltage commands were generated using pCLAMP 8.0 software (Molecular Devices Corp). Specific voltage-clamp protocols are described in the Results section. Currents were not corrected for leak or endogenous currents and capacitance transients were not nulled.

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HEK-293 Cells

The human embryonic kidney cell line (HEK-293) was used for expression of hERG, KCNQ1, and KCNE1 constructs. All cDNAs were kindly given to us by Michael Sanguinetti (University of Utah, Salt Lake City, UT). hERG cDNA was subcloned into pBK-CMV (Stratagene, La Jolla, CA) using HindIII/SmaI sites. KCNQ1 and KCNE1 were subcloned in pCEP4 (Invitrogen, Carlsbad, CA). Cells were grown in 60-mm tissue culture dishes (Corning, Corning, NY) in DMEM with 10% horse serum and 1% antibiotic antimycotic solution (Sigma, Mexico) in a humidified incubator at 37°C (5% CO2) and passaged every 3 to 4 days. For transfection, cells were plated in a culture dish and transfected 24 hours later with Lipofectamine 2000 reagent (Invitrogene, La Jolla, CA) according to the instructions of the manufacturer. For electrophysiological study, the cells were harvested from the culture dish by trypsinization, washed twice with standard DMEM medium, and stored in this medium at room temperature for later use. Cells were studied within 8 hours of harvest.

Voltage-clamp recordings of cells were made with an Axopatch-200B amplifier (Molecular Devices Corp) using the perforated patch-clamp technique.12 Macroscopic current recordings were performed in whole cell configuration of the patch-clamp technique13 at room temperature (21°C-23°C) and were sampled at 1 kHz to 10 kHz after anti-alias filtering at 0.5 kHz to 5 kHz. Data acquisition and command potentials were controlled by pClamp 8.0 software (Molecular Devices Corp). The electrode resistance was 1.5 to 3.0 MΩ after filling with an internal solution that contained 100 mM KCl, 10 mM HEPES, 5 mM K4BAPTA, 5 mM K2ATP, and 1 mM MgCl2, nystatin 200 μg/mL; pH was adjusted to 7.2 with KOH. The bath solution contained 130 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose; pH was adjusted to 7.35 with NaOH.

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Drugs

Berberine (Sigma, St. Louis, MO) was dissolved directly in the external solution at the desired concentrations. Oocytes and HEK-293 cells were exposed to berberine solutions until steady-state effects were achieved, usually in about 15 minutes. To determine the concentration-effect relationships, a single oocyte was exposed to cumulative concentrations of berberine.

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Data Analysis

Data are presented as mean ± SEM (n = number of cells). pClamp 8.0 software (Molecular Devices Corp) was used to perform nonlinear least-squares kinetic analyses of time-dependent currents. The fractional block of current (f) was plotted as a function of drug concentration ([D]) and the data fit with a Hill equation:

to determine the concentration (IC50) required for 50% block of current magnitude and the Hill coefficient, nh. The voltage dependence of hERG and KCNQ1/KCNE1 activation was determined from tail currents measured at −70 mV following 4-second test depolarizations. Normalized tail current amplitude (I n) was plotted versus test potential (Vt) and fitted using Origin software (Northampton, MA) to a Boltzmann function:

V1/2 is the voltage at which the current is half-activated and k is the slope factor of the relationship. Statistical comparisons between experimental groups were performed using ANOVA and Dunnet's method. Differences were considered significant at P < 0.05.

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RESULTS

Effect of Berberine on hERG and KCNQ1/KCNE1 Channels Expressed in HEK-293 Cells

hERG currents were elicited by 2-second depolarizing steps to +20 mV, followed by repolarization to −60 mV. These pulses were repeated every 10 seconds. Figure 1A shows current traces measured under control conditions and during superfusion of the cell chamber with berberine at a concentration of 1, 3, and 10 μM. Currents were inhibited by berberine in a concentration-dependent manner. In addition, berberine induced the tail current crossover (Fig. 1A), suggesting an open channel block and that the drug molecule is bound to the inner pore of the channel preventing channel closure. Unbinding of the drug is required before the channel can close, resulting in a delayed onset of deactivation and hence, the crossover of the macroscopic tail currents.14 The effect of berberine at concentrations 10 μM or lower was completely reversed after washout of the drug during 15 minutes.

FIGURE 1

FIGURE 1

KCNQ1/KCNE1 currents were elicited applying 3-second depolarizing steps to +50 mV, followed by repolarization to −40 mV. The pulses were applied every 10 seconds. Figure 1B shows a current trace measured under control conditions and during superfusion of berberine of 100 μM. Berberine reversibly decreased KCNQ1/KCNE1 currents by 11 ± 4%. Analysis of the concentration-response relationship in hERG channels indicated an IC50 of 3.1 ± 0.5 μM and a Hill coefficient, nH of 0.85 (Fig. 1C). From these experiments is clear that hERG currents are more sensitive to berberine than KCNQ1/KCNE1 currents.

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Block of KCNQ1/KCNE1 and hERG Channels Expressed in Oocytes

We next compared the effects of berberine on KCNQ1/KCNE1 and hERG channels expressed in Xenopus oocytes (Figs. 2 and 3). KCNQ1/KCNE1 currents expressed in Xenopus oocytes were elicited by 3-second depolarization pulses from a holding potential of −80 mV. The test potentials ranged from −20 to +50 mV, and were applied in 10-mV increments every 20 seconds. Tail currents were recorded by return of the membrane potential to −40 mV (Fig. 2A). Berberine 300 μM reduced both the current during the depolarizing pulse and the tail currents (Fig. 2B). In Figure 3C, traces of currents during depolarizing pulses to −10 and +50 mV and tail currents upon return to −40 mV, under control and in the presence of berberine were superimposed. The current amplitude decrease compared with control induced by berberine was significantly greater at −10 than at +50 mV. A plot of the maximum current amplitudes measured at the end of 3-second is shown in Figure 2D and tail currents in Figure 2E. The amplitude of tail current as a function of test potential was fitted with a Boltzmann function; this relationship had a V1/2 of 18.6 ± 2.4 mV and a slope factor (k) of 14.1 ± 1.4 mV in control. In the presence of berberine V1/2 was not significantly different, 22.2 ± 2.9 mV with a k of 15.4 ± 1.5 mV (Fig. 2D). Figure 2F shows the voltage-dependent effect of berberine on KCNQ1/KCNE1 currents. The fractional block measured at the end of the 3-second depolarizing pulse (1 - Idrug/Icontrol) is plotted as a function of the depolarizing pulse membrane potential. The blocking effect of berberine was decreased by membrane depolarization. The effects of berberine on both current measured during the test pulses and tail current amplitudes were completely reversed after 15 minutes washout of drug (Figs. 2D and 2E).

FIGURE 2

FIGURE 2

FIGURE 3

FIGURE 3

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Voltage-Dependent Block of WT hERG Channels Expressed in Oocytes

hERG channels were expressed in Xenopus oocytes and currents were elicited by 4-second depolarization pulses from a holding potential of −80 mV. The test potentials ranged from −70 to +40 mV, and were applied in 10-mV increments every 20 seconds. Tail currents were recorded by return of the membrane potential to −70 mV. WT currents during the depolarizing pulses reached a maximum at −10 mV and declined at more positive potentials (Figs. 3A and 3D). The same oocyte was then treated with 100 μM berberine for 15 minutes and the voltage-clamp protocol was repeated. Berberine reduced both the current during the depolarizing pulse and the tail currents (Fig. 3B and C). In Figure 3C, traces of currents during depolarizing pulses to −40 and +20 mV and tail currents upon return to −70 mV under control and in the presence of berberine were superimposed. It is clear that current amplitude elicited by the depolarizing pulse to −40 and tail current amplitude under control conditions and in the presence of berberine were similar. On the other hand, both, current amplitude during the depolarizing pulse to +20 mV and tail current were significantly decreased by the drug. A plot of the maximum current amplitudes measured at the end of 4-second pulses indicates that block by berberine was voltage-dependent with more pronounced reductions in current at more depolarized test potentials (Fig. 3D). The decrease in tail current by berberine exhibited a similar voltage dependency (Fig. 3E). The amplitude of tail current as a function of test potential was fitted with a Boltzmann function. This relationship had a V1/2 of -24.7 ± 2.8 mV and a k of 9.7 ± 0.8 mV. In the presence of berberine, the V1/2 was shifted slightly, to -29.8 ± 2.7 with no change in k (9.6 ± 1.0 mV). The effects of berberine on both current measured during the test pulses and tail current amplitudes were completely reversed after 15 minutes washout of drug (Figs. 3D and 3E). The voltage dependence of WT hERG channel tail current block is quantified in Figure 3F. The fractional block measured at the end of the 4-second depolarizing pulses (1 - Idrug/Icontrol) was plotted as a function of the depolarizing pulse membrane potential. The blocking effects of berberine were increased by membrane depolarization.

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Characterization of the Berberine Binding Site

Several residues located on the S6 domain and the pore helix of hERG comprise the putative binding site for methanesulfonanilides, vesnarinone, chloroquine, and quinidine.9,15-17 Mutation of V625 of the pore helix, or Y652 or F656 of the S6 domain to Ala caused the most profound reductions in potency for block of hERG current by MK-499.9 Therefore, we determined the concentration-effect relationship for berberine on WT, V625A, Y652A, and F656T hERG channels expressed in oocytes, measured as peak tail current at −70 mV after a 4-second pulse to 0 mV.

The effects of 100 μM berberine on WT hERG and 300 μM on Y652A, F656T, and V625A hERG are shown in Figure 4A through 4D. The concentration-response relationship for block by berberine of WT hERG currents expressed in Xenopus oocytes is shown in Figure 4E. The IC50 was 80 ± 5 μM with a Hill coefficient (nH) of 1.04. As reported previously,9 tail currents for V625A hERG channels were inward at −70 mV due to a decrease in K+ selectivity. The expression of F656A current was low, and the biophysical properties of the channel different than WT hERG.9 Therefore, in the present work we used F656T hERG, a channel that expresses well and retains relatively normal biophysical properties.18 The IC50 for berberine was estimated by extrapolation to be about 730 ± 65 μM for V625A. At the highest concentration used (300 μM), the drug did not significantly affect Y652A and F656T hERG channels (Fig. 4E). Thus, Y652A and F656T mutations caused a dramatic reduction in drug potency, whereas the mutation V625A reduced potency by a factor of about 10.

FIGURE 4

FIGURE 4

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Voltage-Dependent Block of Y652 Mutant hERG Channels

We previously reported that mutation of Y652 to Ala (Y652A) inverted the voltage dependence of channel block by quinidine or chloroquine compared with WT hERG.16,17 These drugs reduced WT hERG more as the membrane was progressively depolarized, whereas Y652A channels were less blocked by increasing membrane depolarization. In contrast, mutation of Y652 to Phe (Y652F) induced a voltage-independent block by quinidine and chloroquine.17 As shown in Figures 5 and 6, mutations Y652A and Y652F had similar effects on the voltage-dependent block of hERG by berberine. The effect of berberine on Y652A hERG channel current elicited at different test potentials are shown in Figures 5A through 5C. The amplitude of tail current as a function of test potential was fit with a Boltzmann function and had a V1/2 of -15.4 ± 1.8 mV and a k of 9.9 ± 0.8 mV in control (Fig. 5D). In the presence of berberine, the voltage dependence of activation was not significantly changed (V1/2 = -13.5 ± 1.7 mV; k = 9.4 ± 1.0 mV). A plot of the fractional current block (1 - Idrug/Icontrol) at different membrane potentials (Fig. 5E) indicates that block by berberine was voltage-dependent with less pronounced reductions in current at more depolarized test potentials. Thus, the voltage dependence for block of Y652A hERG was inverted compared with WT hERG, similar to our previous findings with chloroquine and quinidine.

FIGURE 5

FIGURE 5

FIGURE 6

FIGURE 6

Berberine did not significantly modify the V1/2 of the activation curve for Y652F from −33.4 mV under control to −36.3 mV in the presence of berberine, 10 μM (Fig. 6). Fractional block (1 - Idrug/Icontrol) of Y652F channels was weakly voltage-dependent (Fig. 6E).

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Voltage-Dependent Block of V625A Mutant hERG Channel

The effect of berberine on V625A hERG channel, evaluated with the tail currents measured at −70 mV after an activation pulse elicited at different test potentials in the same cell are shown in Figures 7A and 7B. A plot of the tail current amplitudes indicates that block by berberine was voltage dependent with less pronounced reductions in current at more depolarized test potentials (Figs. 7C and 7D). The amplitude of tail current as a function of test potential was fit with a Boltzmann function; this relationship had a V1/2 of -28.5 ± 1.1 mV and a k of 8.9 ± 0.7 mV in control. In contrast to results obtained in WT hERG channels, berberine appeared to shift the voltage dependence of V625A hERG channels to more positive potentials. In the presence of berberine V1/2 was shifted to -17.1 ± 0.9 mV with a k of 10.7 ± 1.1 mV.

FIGURE 7

FIGURE 7

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Block of Inactivation-Deficient hERG Channels by Berberine

The hypothesis that berberine might preferentially bind to inactivated channels was tested with a hERG channel containing 2 point mutations (G628C/S631C) that eliminates inactivation.19 Unlike WT hERG, whole cell current conducted by these inactivation-deficient channels was progressively increased by greater depolarizations of the membrane (Fig. 8A). However, similar to WT hERG, the block of current conducted by mutant channels was increased at more positive membrane potentials (Fig. 8B and 8C). For example, the decrease of G628C/S631C hERG current was 31% at −40 mV and was increased to 47% at +20 mV (Fig. 8D).

FIGURE 8

FIGURE 8

The concentration-effect of berberine on G628C/S631C hERG channels was assessed by repetitively applying 4-second pulses to 0 mV at a frequency of 0.1 Hz from a holding potential of −80 mV (Fig. 8E). The relative inhibition of current is plotted as a function of berberine concentration (Fig. 8F). The IC50 for G628C/S631C hERG channels was 113 ± 7.7 μM (nH = 1.08 ± 0.08). Thus, removal of inactivation did not significantly decrease the potency of berberine, compared with the IC50 obtained on WT channels (Fig. 4E), indicating that the drug can readily bind to open channels and that inactivation is not required for block.

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DISCUSSION

In the present work, we characterized the effects of berberine on hERG and KCNQ1/KCNE1 channels heterologously expressed in HEK-293 cells and Xenopus oocytes. The effect of berberine on hERG expressed in HEK-293 cells was similar to the effects of berberine on IKr of feline ventricular myocytes,3 corroborating that the main potassium channel target of berberine in cardiac myocytes is IKr. On the other hand, 100 μM berberine only inhibited KCNQ1/KCNE1 channels heterologously expressed in HEK-293 cells by 11%. Wang and Zheng4 reported in guinea pig ventricular myocytes that berberine (30 μM) did not affect IKr, but, at the same concentration it decreased IKs, 34%. The effects of berberine on both channels were reversible after washout of the drug.

The effect of berberine on hERG expressed in both heterologous systems was more potent than the effect of the drug on KCNQ1/KCNE1. As observed with other drugs,20 the effect of berberine on hERG expressed in HEK-293 cells was more potent (IC50, 3.1 μM) than on hERG expressed in oocytes (IC50, 80 μM, berberine 300 μM). This difference has been found with most drugs and has been attributed to the presence of yolk inside the oocytes, which can absorb the drug and lower the effective intracellular free drug concentration.20

Understanding the molecular determinants of drug binding to hERG channels is important for design of new drugs devoid of side effects like the acquired long QT syndrome. Previous studies defined residues important for drug binding by using a site-directed mutagenesis approach.9,21 Two residues (Y652 and F656) located in the S6 domain and a residue located at the base of the pore helix (V625A) that face the central cavity of the channel appear to be essential components of the hERG binding site for high and low potency drugs alike, including MK-499, dofetilide, vesnarinone, and quinidine.9,15,16 However, it was shown that F656A and Y652A mutations in hERG only partially attenuated block by fluvoxamine and dronedarone.22,23 These results indicate that not all drugs bind to the same site, and there is not a universal binding site for the different hERG channel blockers. In the present work, we have confirmed the importance of V625, Y652, and F656 as sites of interaction for yet another drug, berberine.

Block of WT hERG by berberine was found to be voltage dependent, with enhanced block in response to increasing membrane depolarization. The voltage-dependent component of block by berberine can be reversed by mutation of Y652 to Ala and eliminated by mutation to Phe. Similar results were described for the quinolines chloroquine, quinidine, and quinine.16,17 We previously suggested that this voltage-dependent block results from gating dependent changes in the accessibility of Y652, a critical component of the drug binding site. Positive transmembrane potentials may increase the accessibility of Y652, resulting in more potent block. We have also found that berberine block of the hERG V625A mutant was decreased by membrane depolarization, suggesting that the second configuration of the berberine binding site in addition to the Y652 residue involves V625.

Previous studies have provided evidence that binding to hERG channels requires or is greatly enhanced by intact C-type inactivation because many mutations that disrupt inactivation dramatically reduce the sensitivity to methanesulfonanilides24 and even low-potency drugs like d-sotalol.25 These findings suggest that allosteric changes in channel configuration associated with inactivation gating induce modifications in the binding site for some drugs. However, in the present work we found that the non inactivating G628C/S631C and WT hERG channels have similar sensitivity to block by berberine, indicating that this drug is an open channel blocker that does not require inactivation to achieve normal levels of block. A similar conclusion was reached for block of hERG by low affinity drugs such as disopyramide,26 quinidine,21 vesnarinone,15 and chloroquine.16

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CONCLUSION

In conclusion, berberine blocked hERG currents expressed in HEK-293 cells at similar concentrations that blocked IKr and increased action potential duration in cat ventricular myocytes.3 Berberine also blocked KCNQ1/KCNE1 currents with a lower potency than hERG. Thus, our results confirm that IKr is an important target for the cardiac electrophysiologic effects of berberine. Three residues that face the central cavity of the channel (V625, Y652, and F656) appear to be essential components of the hERG binding site for berberine.

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ACKNOWLEDGMENTS

The authors thank Marcelo Montes de Oca and Peter Westenskow for technical assistance. We also thank Dr. Michael C. Sanguinetti for support and critical reading of the manuscript.

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REFERENCES

1. Lau CW, Yao XO, Ko WH, et al. Cardiovascular actions of berberine. Cardiovasc Drug Rev. 2001;19:234-244.
2. Ricciopo Neto F. Electropharmacological effects of berberine on canine cardiac Purkinje fibres and ventricular muscle and atrial muscle of the rabbit. Br J Pharmacol. 1993;108:534-537.
3. Sanchez-Chapula J. Increase in action potential duration and inhibition of the delayed rectifier outward current IK by berberine in cat ventricular myocytes. Br J Pharmacol. 1996;117:1427-1434.
4. Wang YX, Zheng YM. Ionic mechanism responsible for prolongation of cardiac action potential duration by berberine. J Cardiovasc Pharmacol. 1997;30:214-222.
5. Sanguinetti MC, Jiang C, Curran ME, et al. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell. 1995;81:299-307.
6. Barhanin J, Leasage F, Guillemare E, et al. KvLQT1 and IsK (mink) proteins associate to form the IKs potassium channel. Nature. 1996;384:78-80.
7. Sanguinetti MC, Curran ME, Zou A, et al. Coassembly of KvLQT1 and minK (IsK) proteins to form IKs potassium channel. Nature. 1996;384:80-83.
8. Ficker E, Jarolimek W, Brown AM. Molecular determinants of inactivation and dofetilide block in ether-a-go-go (EAG) channels and EAG-related channels. Mol Pharmacol. 2001;60:1343-1348.
9. Mitcheson JS, Chen J, Lin M, et al. A structural basis for drug-induced log QT syndrome. Proc Natl Acad Sci USA. 2000;97:12329-12333.
10. Sanguinetti MC, Xu QP. Mutations of the S4-S5 linker alter activation properties of HERG potassium channels expressed in Xenopus oocytes. J Physiol. 1999;514:667-675.
11. Stuhmer W. Electrophysiological recording from Xenopus oocytes. Methods in Enzymology, Ion Channels. 1992;207:319-339.
12. Horn R, Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988;92:145-159.
13. Hamill OP, Marty A, Neher E, et al. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.
14. Armstrong CM. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J Gen Physiol. 1971;58:413-437.
15. Kamiya K, Mitcheson JS, Yasui K, et al. Open channel block of HERG K+ channels by vesnarinone. Mol Pharmacol. 2001;60:244-253.
16. Sanchez-Chapula JA, Navarro-Polanco RA, Culberson C, et al. Molecular determinants of voltage dependent HERG K+ channel block. J Biol Chem. 2002;277:23587-23595.
17. Sanchez-Chapula JA, Ferrer T, Navarro-Polanco RA, et al. Voltage-dependent profile of human ether-a-go-go-related gene channel block is influenced by a single residue in the S6 transmembrane domain. Mol Pharmacol. 2003;63:1051-1058.
18. Fernandez D, Ghanta A, Kauffan GW, et al. Physicochemical features of the HERG channel drug binding site. J Biol Chem. 2003;279:10120-10127.
19. Smith PL, Baukrowitz T, Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature. 1996;379:833-836.
20. Witchel HJ, Milnes JT, Mitcheson J, et al. Troubleshooting problems with in vitro screening of drugs for QT interval prolongation using HERG K+ channels expressed in mammalian cell lines and Xenopus oocytes. J Pharmacol Toxicol Methods. 2002;48:65-80.
21. Lees-Miller JP, Duan Y, Teng GQ, et al. Molecular determinant of high-affinity dofetilide binding to HERG1 expressed in Xenopus oocytes: involvement of S6 sites. Mol Pharmacol. 2000;57:367-374.
22. Milnes JT, Crociani O, Arcangeli A, et al. Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S& mutations at F656 or Y652. Br J Pharmacol. 2003;139:887-898.
23. Ridley JM, Milnes JT, Witchel HJ, et al. High affinity HERG K+ channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656. Biochem Biophys Res Commun. 2004;325:883-891.
24. Ficker E, Jarolimek W, Kiehn J, et al. Molecular determinants of dofetilide block of HERG K+ Channels. Circ Res. 1998;82:386-395.
25. Numaguchi H, Mullins FM, Johnson JP, et al. Probing the interaction between inactivation gating and D-sotalol block of HERG. Circ Res. 2000;87:1012-1018.
26. Paul AA, Witchel HJ, Hancox JC. Inhibition of HERG potassium channel current by the class 1a antiarrhythmic agent disopyramide. Biochem Biophys Res Commun. 2001;280:1243-1250.
Keywords:

berberine; HEK-293; hERG; KCNQ1/KCNE1; IKr; IKs; oocytes

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