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Am J Physiol Heart Circ Physiol 284: H256-H267, 2003. First published October 3, 2002; doi:10.1152/ajpheart.00220.2002
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Vol. 284, Issue 1, H256-H267, January 2003

Functional and pharmacological properties of canine ERG potassium channels

Jixin Wang, Kimberly Della Penna, Hao Wang, Jerzy Karczewski, Thomas M. Connolly, Kenneth S. Koblan, Paul B. Bennett, and Joseph J. Salata

Department of Molecular Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We established HEK-293 cell lines that stably express functional canine ether-à-go-go-related gene (cERG) K+ channels and examined their biophysical and pharmacological properties with whole cell patch clamp and 35S-labeled MK-499 ([35S]MK-499) binding displacement. Functionally, cERG current had the hallmarks of cardiac delayed rectifier K+ current (IKr). Channel opening was time- and voltage dependent with threshold near -40 mV. The half-maximum activation voltage was -7.8 ± 2.4 mV at 23°C, shifting to -31.9 ± 1.2 mV at 36°C. Channels activated with a time constant of 13 ± 1 ms at +20 mV, showed prominent inward rectification at depolarized potentials, were highly K+ selective (Na+-to-K+ permeability ratio = 0.007), and were potently inhibited by IKr blockers. Astemizole, terfenadine, cisapride, and MK-499 inhibited cERG and human ERG (hERG) currents with IC50 values of 1.3, 13, 19, and 15 nM and 1.2, 9, 14, and 21 nM, respectively, and competitively displaced [35S]MK-499 binding from cERG and hERG with IC50 values of 0.4, 12, 35, and 0.6 nM and 0.8, 5, 47, and 0.7 nM, respectively. cERG channels had biophysical properties appropriate for canine action potential repolarization and were pharmacologically sensitive to agents known to prolong QT. A novel MK-499 binding assay provides a new tool to detect agents affecting ERG channels.

ion channels; acquired long QT syndrome, potassium ion channel; ventricular arrhythmia; canine ether-à-go-go-related gene


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ACQUIRED long QT syndrome occurs commonly as a result of drug inhibition of IKr, the rapid delayed rectifier K+ current in human ventricular myocardium. The human ether-à-go-go-related gene (herg) K+ channel protein (hERG) (29, 35) underlies IKr and plays a critical role in human myocardial repolarization (22, 23). Mutations in herg are associated with both the congenital (LQT2; inherited) and the acquired (aLQT; drug induced) long QT syndrome and attendant fatal cardiac arrhythmias (19).

Drug safety is a critical issue in the development of novel human therapeutic agents, and it is increasingly recognized that new and existing drugs have the potential to induce aLQT through unanticipated inhibition of hERG K+ channels. During the course of drug development, the safety of new drug candidates is addressed in several nonhuman models before human exposure. Canine models are important because of similarities in heart size and the vast knowledge of canine physiology. In vitro canine cardiac tissues are used to study drug effects on action potential morphology and refractoriness. In vivo canine models are used to assess the propensity of drugs to affect the electrocardiogram including the QT interval, an index of myocardial repolarization. However, the biophysics and pharmacology of canine ERG (cERG) are unknown. The purpose of this study was to define the functional and pharmacological properties of cERG heterologously expressed in HEK-293 cells.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Molecular Biology

The cERG cDNA was provided by Dr. Joerg Zehelein (University Hospital, Heidelberg, Germany; Ref. 36). cERG was subcloned into pcDNA3 (Invitrogen) between the HindIII and EcoRI sites downstream of the cytomegalovirus promoter to generate an expression vector, pcDNA3-cERG.

Cell Culture

HEK-293 cells were purchased from American Type Culture Collection and cultured in modified minimum essential medium (MEM-alpha M) supplemented with 10% fetal bovine serum and penicillin-streptomycin (GIBCO). HEK-293 cells were transfected with pcDNA3-cERG with the use of FuGENE 6 transfection reagent (Roche). Selection started 2 days after transfection in medium containing 800 µg/ml geneticin (G-418; GIBCO). After 3 wk of selection, single colonies were cloned with cloning cylinders. A total of 13 cloned colonies were scaled up and maintained in selection medium with 100 nM astemizole (Sigma). HEK-293 cells stably transfected with herg were obtained from Drs. Craig January and Gail Robertson (University of Wisconsin, Madison, WI; Ref. 37).

Western Blot Analysis

For each cell line, 3 × 105 cells were seeded in parallel 35-mm dishes and grown overnight to similar levels of confluence. The next day the medium was removed by aspiration, and the cells were washed twice with PBS, then directly lysed with 0.5 ml of Laemmli sample buffer (Bio-Rad) containing 1 µl of protease inhibitor cocktail (Sigma). One-twentieth of the cell lysate from each sample was boiled for 5 min and loaded onto a Novex NuPAGE 3-8% Tris-acetate gel (Invitrogen/Novex) and then electrophoretically transferred onto a 0.45-µm nitrocellulose membrane. Western blot analysis was performed with the WesternBreeze chemiluminescent immunodetection system (Invitrogen/Novex) according to the manufacturer's instructions, and antibody specific to hERG (anti-hERG, APC-016; Alomone Labs) at a 1:200 dilution was used to detect the cERG and hERG proteins.

Immunostaining

The expression of cERG in the cells was detected by immunostaining with primary antibodies (anti-hERG, APC-016; Alomone Labs) as described previously (32). Cells were plated at 40,000 cells/well onto a 16-chamber slide coated with poly-D-lysine and grown overnight to confluence. Cells were first fixed with 0.2 ml of cold 4% paraformaldehyde for 20 min and then washed four times with cold PBS. After incubation in blocking buffer (3% BSA, 3% normal goat serum, 0.1% Triton X-100 in PBS) for 1 h, primary antibody (anti-hERG) diluted 1:100 in blocking buffer was added to the cells and incubated for 1 h at room temperature. After being washed with PBS, secondary antibody (goat anti-rabbit IgG Cy3; Jackson Immunoresearch) diluted 1:500 in blocking buffer was added to the cells and incubated for 30 min at room temperature. The slides were washed six times with PBS, dried, and covered with Vectashield mounting medium with DAPI (Vector Labs).

Cell Preparation for Electrophysiology

HEK-293 cERG cell lines were grown to 80% confluence, washed two times with PBS, trypsinized with 0.05% trypsin-EDTA (GIBCO) for 2 min at 37°C, and resuspended in culture medium. Cells were studied within 8 h.

Voltage-Clamp Recording Methods

Cells for electrophysiological study were transferred to an 80-µl recording chamber (RC-24; Warner Instrument) and superfused with a solution containing (in mM) 132 NaCl, 4 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 HEPES, and 11 glucose, pH 7.2, at a flow rate of 0.8 ml/min, allowing rapid solution changes. The temperature was maintained at 36 ± 1°C with a water-jacketed preheating system and a DC-powered heating system (Cell MicroControls, Virginia Beach, VA). In some experiments, currents were recorded at room temperature of 23 ± 1°C. K+ currents were recorded with the whole cell patch-clamp technique. An Axopatch 200A patch-clamp amplifier was connected to a Pentium PC computer (Compaq Deskpro 6000) through a Digidata 1200 interface (Axon Instruments, Foster City, CA). Patch pipettes were fabricated from capillary glass obtained from Kimble Products (0.8-1.1 × 100 mm, no. Kimax-51) with a vertical micropipette puller (model L/M-3p-A, List-Medical). Pipettes resistances were 2-4 MOmega when filled with a solution containing (in mM) 119 K-gluconate, 15 KCl, 3.2 MgCl2, 5 EGTA, 5 K2ATP, and 5 HEPES, pH 7.35. Cell capacitance and series resistance were compensated (80-90%) before recording. Data were acquired and analyzed with pCLAMP 8 (Axon Instruments), and results were plotted with Origin (Microcal Software, Northampton, MA).

Voltage-Clamp Data Analysis

Channel kinetics were evaluated by fitting time (t)-dependent currents appropriately normalized with exponential functions consisting of one or more terms
y(t)=A<SUB>0</SUB><IT>+</IT><LIM><OP>∑</OP><LL><IT>i</IT></LL><UL><IT>n</IT></UL></LIM> A<SUB><IT>i</IT></SUB> exp<SUP>−<IT>t/&tgr;<SUB>i</SUB></IT></SUP> (1)
where i = 1 or 2 and Ai and tau i represent the amplitude and time constants of the ith component, respectively. The fits were based on nonlinear least-squares methods. The goodness of fit was determined by examining the residuals and visual inspection. Voltage dependence of channel opening was estimated by fitting a form of a Boltzmann equation
I/I<SUB>max</SUB><IT>=</IT>[1<IT>+</IT>exp(<IT>V</IT><SUB>m</SUB><IT>−V</IT><SUB>½</SUB>)<IT>/k</IT>]<SUP><IT>−</IT>1</SUP> (2)
where Vm represents membrane potential, V1/2 is the membrane potential when tail current (I) normalized to maximum current amplitude (Imax) (I/Imax) = 0.5, and k is a slope factor. Alternatively, this equation can be written in terms of thermodynamic parameters
I/I<SUB>max</SUB><IT>=</IT>[(1<IT>+</IT>exp<SUP><IT>&Dgr;</IT>G<SUB>0</SUB><IT>+</IT>(<IT>V</IT><SUB>m</SUB><IT> z &dgr;e/K</IT><SUB>b</SUB><IT> · </IT>T)</SUP>)]<SUP><IT>−</IT>1</SUP> (3)
where Delta G0 represents a non-voltage-dependent free energy term, z represents the number of gating charges, delta  is the fraction of the electrical field, e is the fundamental charge of an electron, and Kb and T are the Boltzmann constant and absolute temperature, respectively (1).

Ionic selectivity of the cERG channels was determined by examining the reversal potential (Erev) at various extracellular K+ concentrations ([K+]o). Data were fit with the Goldman-Hodgkin-Katz equation
E<SUB>rev</SUB><IT>=</IT>2.303<FR><NU>RT</NU><DE><IT>F</IT></DE></FR>log<SUB>10</SUB><FR><NU>[K]<SUB>o</SUB> + &agr;[Na]<SUB>o</SUB></NU><DE>[K]<SUB>i</SUB> + &agr;[Na]<SUB>i</SUB></DE></FR>; &agr; = <FR><NU><IT>P</IT><SUB>Na</SUB></NU><DE><IT>P</IT><SUB>K</SUB></DE></FR> (4)
where R is the gas constant, T is temperature, F is the Faraday constant, [K+]i and [Na+]i are intracellular K+ and Na+ concentrations, respectively, [Na+]o is extracellular Na+ concentration, and PNa/PK is the best-fit permeability ratio of Na+ to K+.

MK-499 Binding Assay

Cell membranes were prepared from HEK-293 cells constitutively expressing hERG or cERG. Cells were harvested and homogenized in Tris-EDTA buffer containing 50 mM Tris and 1 mM EDTA, pH 7.4. Homogenates were centrifuged at 45,000 g for 20 min at 4°C. The pellet was washed once in Tris-EDTA, centrifuged, resuspended at a concentration of 5 mg/ml in binding buffer containing (in mM) 71.5 NaCl, 60 KCl, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.4, and stored at -70°C.

On the day of assay, cERG or hERG membranes were thawed and diluted to a concentration of 10 µg/ml in binding buffer and 35S-labeled MK-499 ([35S]MK-499) was added to achieve a final concentration of 50 pM. A 400-µl aliquot of this solution was added to each well of a 96-well plate. For competition binding studies, test compounds in DMSO at varying concentrations or DMSO alone (control) were added such that the final DMSO concentration was 1%. After incubation for 2 h at room temperature, the binding was stopped by filtration of membranes through 96-well GF/B Unifilters (Packard). The filters were washed with 3 ml of ice-cold wash buffer containing (in mM) 131.5 NaCl, 1 CaCl2, 2 MgCl2, and 10 HEPES, pH 7.4, filters were dried, and radioactivity associated with each filter was measured by the addition of Microscint-20 scintillation liquid and counting in a 96-well scintillation counter (Topcount, Packard).

This filtration method is valid for this radioligand. The dissociation of [35S]MK-499 occurred with a half time (T1/2) of ~45 min and a first-order rate constant (koff) of ~2.57 × 10-4 s-1, at room temperature. As a general rule of thumb, for compound with a Kd of ~1 nM, >90% remains bound at 1.7 min (8). From our off-rate data, it is estimated that >98% of the [35S]MK-499 remains bound at 1 min. Filtration and wash steps were completed within 1 min, and washing was done with ice-cold buffer to further slow ligand dissociation.

Pharmacological Data Analyses and Statistics

Data are expressed as means ± SE. Group means were compared with an unpaired t-test, and differences were considered significant at the P < 0.05 level. Drug concentrations producing 50% inhibition (IC50) of K+ current or [35S]MK-499 binding were determined by fitting of a Hill equation to the concentration response data
% inhibition = 100 − <FR><NU>100</NU><DE>1 + ([drug]/IC<SUB>50</SUB>)<SUP><IT>n</IT><SUB>H</SUB></SUP></DE></FR> (5)
where nH is the Hill coefficient.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Western Blot Analysis and Immunostaining of cERG Expressed in HEK-293 Cells

The individual clonal cells were screened initially for expression of cERG protein by Western blot analysis and immunostaining. With these methods, three cell lines that expressed high levels of cERG channel protein were selected and evaluated further with voltage clamp. All three selected lines had similar current densities and properties. One particular line (HEK-293 cERG no. 2) was chosen and used exclusively in this study for characterization of the functional and pharmacological properties of the expressed cERG current.

Western blot analysis of total protein was conducted on three HEK-293 cell lines: nontransfected (control), hERG stably transfected, and cERG stably transfected (Fig. 1). Antibodies directed against the COOH terminus of hERG were used to probe the hERG and cERG protein. The antibodies recognized two bands of 155 and 135 kDa in HEK-293 hERG lines. The cERG and hERG cells displayed very similar bands consistent with the predicted identical size of the hERG and cERG proteins.


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  		Fig. 1.   Expression of canine ether-à-go-go-related gene protein (cERG) in HEK-293 cells as detected by Western blot analysis. Two major bands are apparent at ~135 and 155 kDa, presumably corresponding to core-glycosylated and fully glycosylated forms of the cERG protein, respectively. hERG, human ERG.

Immunostaining of the cell lines detected cERG protein expression in HEK-293 cERG cells. The protein level appeared especially high in the cERG cell membranes (Fig. 2A). A similar staining pattern was seen in HEK-293 hERG cells (Fig. 2B) but not in the HEK-293 control cells (Fig. 2C).


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  		Fig. 2.   Expression of cERG and hERG as detected by immunostaining. A: HEK-293 cERG. B: HEK-293 hERG. C: HEK-293 nontransfected control cells.

Biophysical Properties of cERG K+ Current

Membrane potential-dependent channel activation. Figure 3 shows voltage-dependent properties of cERG current at 23°C. Figure 3A illustrates superimposed whole cell K+ currents recorded during voltage-clamp steps to different potentials. The holding potential was -80 mV; the cell was depolarized to membrane potentials ranging between -60 and +50 mV for 2 s to activate cERG current, and the cell was then clamped to -50 mV to record deactivating tail currents. During depolarizing steps, an outward current was activated at voltages positive to -40 mV and the current amplitude increased to a maximum at +10 mV. With increasing depolarization, the current amplitude decreased progressively. Outward deactivating K+ tail currents were observed during a return to -50 mV. Figure 3B plots the outward K+ current amplitude at the end of each depolarizing step. The apparent inward rectification has been commonly observed in hERG channels and is attributed to rapid voltage-dependent channel inactivation (5, 22, 24, 27, 29). Normalized tail current amplitudes were used to construct voltage-activation curve as shown in Fig. 3B. The threshold voltage for opening or activation of cERG channels was near -50 mV, and channels were fully activated at membrane potentials near 0 mV. The averaged peak tail current density at a membrane potential of +40 mV was 55 ± 16.7 pA/pF at 23°C. The averaged half-maximum activation voltage (V1/2) and slope factor k derived by fitting a Boltzmann function (Eq. 2) to the data at 23°C were -7.8 ± 2.4 and 8.2 ± 0.7 mV, respectively (see Table 1).


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  		Fig. 3.   Voltage-activation relationship of cERG channels stably expressed in HEK-293 cells. A: example current traces elicited from a holding potential (Vh) of -80 mV with 2-s depolarizing steps to varying test voltages (Vt) between -60 and +50 mV in 10-mV increments at 23°C. Inset: voltage-clamp protocol. B: plots of the normalized current density (pA/pF) relations for the peak tail current on repolarization (Vtail) to -50 mV and time-dependent current measured at the end of the test pulse for the cell in A. Data are normalized to cell capacitance. Note that the time-dependent current displays prominent inward rectification at Vt greater than or equal to +10 mV, characteristic of native delayed rectifier K+ current and expressed hERG current.


                              
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  		Table 1.   Voltage dependence of activation and ionic selectivity of cERG compared with hERG channels expressed in HEK-293 cells

Kinetics of cERG activation gating: effect of temperature. The properties of channels measured at 36°C are more representative of native channel behavior under normal physiological conditions. Most experiments reported here were conducted at 36°C. Figure 4A shows an example of the effect of temperature on cERG K+ currents. The channels were forced to open (activated) by a 4-s step to 0 mV and then closed again by stepping to -50 mV. The ensemble kinetics of channel activation at 0 mV and the kinetics of channel closing at -50 mV can be seen. Increasing the temperature from 23°C to 36°C resulted in several changes. There was a marked increase in the amplitude of the K+ current. This coincided with a prominent acceleration of the rate of activation during the depolarization to 0 mV as well as an increase in the ensemble average kinetics of channel closing at -50 mV. The increase in K+ current at 36°C can be attributed in large part to the accelerated kinetics of channel opening (Table 2). Depending on test voltage, a single- or double-exponential function (Eq. 1) was best fit to the activation and deactivation of the cERG current. At comparable voltages, elevating the temperature decreased both the fast (tau f) and slow (tau s) time constants of activation and deactivation and increased the relative contribution of tau f to each. For example, at a test potential of -20 mV, the averaged tau f and tau s of activation were 284 and 3,772 ms at 23°C, respectively, and these were decreased to 128 and 664 ms, respectively, at 36°C. In this instance, the fractional contribution of tau f to the activation process [Af/(Af + As)] increased from 0.15 to 0.43 (n = 4). Likewise, at -50 mV, the averaged tau f and tau s of deactivation were 440 and 2,075 ms, respectively, at 23°C and were decreased to 109 and 653 ms, respectively, at 36°C, whereas [Af/(Af + As)] increased from 0.40 to 0.62 (n = 4). A plot of the effects of temperature on the voltage-activation curve is shown in Fig. 4B. Cooling from near body temperature (36°C) to room temperature (23°C) stabilized the closed state of the channel by ~18 kJ/mol, shifting the V1/2 from -31.9 to -7.8 mV, while k increased slightly from 6 to 8.2 mV (Table 1).


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  		Fig. 4.   Temperature dependence of cERG activation. A: example cERG current traces recorded from a HEK-293 cell at 23°C and 36°C. Currents were elicited from a Vh of -80 mV with a 4-s depolarizing step to a Vt of 0 mV. Repolarizing tail current was measured at -50 mV. B: plots of the voltage dependence of activation. Peak amplitude of deactivating tail currents were measured at -50 mV after 4-s steps to Vt ranging from -60 to +50 mV. Tail currents were normalized to maximum current amplitude for each cell. A Boltzmann function (Eq. 3, MATERIALS AND METHODS) was fit to the averaged data with a nonlinear least-squares fitting routine. T, absolute temperature; triangle triangle G, free energy.


                              
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  		Table 2.   Temperature dependence of kinetics of activation and deactivation of cERG

A quantitative analysis of the time courses of cERG activation and deactivation is displayed in Fig. 5, and the derived time constants are listed in Table 3. The activation time courses between -40 and +60 mV were measured by two methods. First, at membrane potentials <= 0 mV, the rising phase of the K+ current traces were fit directly with a double-exponential function (22, 25) as shown in Fig. 5A. At membrane potentials >0 mV, activating currents were not adequately described by a simple exponential function, most likely because of coincident rapid inactivation (10, 24, 27, 33). Consequently, the time courses of activation at membrane potentials >= 0 mV were estimated by an "envelope of tails" method (16). In an effort to kinetically separate these two processes, an envelope of tail currents was obtained with a voltage-clamp pulse protocol consisting of depolarizing steps applied for varying durations, followed by a repolarizing step to -50 mV to record K+ current tails. The peak amplitude of the K+ current tail is proportional to the number of channels open just before the voltage jump. On repolarization, channels rapidly recover from inactivation; hence, the superimposition of tail current amplitudes determined after voltage steps of varying durations maps the time course of channel opening at any given depolarized membrane potential. A total of 23 superimposed K+ current traces are shown in Fig. 5B. Each successive record was obtained with an incrementally longer depolarizing voltage-clamp step to 0 mV to map the kinetics. The peak of each tail current and a single-exponential fit are shown in Fig. 5A. The tau f derived by fitting either the activating current or the envelope of tails for a test pulse to 0 mV were in close agreement, 39.9 and 31.5 ms, respectively.


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  		Fig. 5.   Time course of cERG current activation and deactivation. A: time course of activation was measured by 2 methods at Vt >=  0 mV, by fitting both the activating current and the envelope of tail currents during a given depolarizing voltage step. Determination of activation time constants (tau ) used a single-exponential equation to fit (solid lines) both the current trace (diamond ) and peaks of the tail currents (open circle ) during a step to a Vt of 0 mV. In this example tau  = 39.9 and 31.5 ms, respectively. B: envelope of tail currents was generated by varying the duration of the voltage step from a Vh of -80 mV to Vt = 0 mV and measuring the peak tail current on repolarization to -60 mV. C: deactivation time course on return to a Vtail of -70 mV after a depolarizing voltage step to a Vt of +60 mV (see also Fig. 7), also best defined by fitting a dual-exponential equation (solid line) to the deactivating current trace (triangle ). D: plots of activation (n = 4-8) and deactivation (n = 7) time constants vs. test voltages. At Vt < 0 mV the activation time course was best defined by fitting a double-exponential equation to the activating current traces, thereby generating two activation tau  values. Data were obtained at 36°C.


                              
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  		Table 3.   Voltage dependence of kinetics of activation and deactivation of cERG at 36°C

The rate of channel closing (deactivation) was determined by fitting the K+ current tails with a double-exponential function. This is exemplified in Fig. 5C, which shows a cERG K+ current tail recorded at -70 mV after an initial depolarizing step to +60 mV for 1 s. The voltage dependence of the tau f and tau s of deactivation are summarized in Table 3 along with the relative amplitudes [Af/(Af + As)] of the two components derived from the curve fitting. The data show that both the fast and slow components of deactivation were highly voltage dependent and that the fast component was greater at more negative membrane potentials. In contrast, the slow component was dominant at more positive membrane potentials. A summary of the time constants derived in this manner from five to eight cells is plotted in Fig. 5D, and the statistical averages are provided in Table 3. The time constants were voltage dependent, ranging from ~20 ms at -80 mV to an average of 79 ms at -40 mV. At more positive membrane potentials up to +60 mV the time constants decreased further to ~10 ms (Table 4; Refs. 25, 37).

                              
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  		Table 4.   Voltage dependence of kinetics of activation and deactivation of hERG at 35°C

cERG inactivation and recovery from inactivation. hERG channels undergo rapid voltage-dependent inactivation at depolarized membrane potentials and recover rapidly on repolarization (24, 27). We tested for these characteristics in cERG channels with a three-pulse protocol (shown in Fig. 6A) to measure the rate of channel inactivation and a two-pulse protocol (Fig. 6B) to measure recovery from inactivation. The three-pulse voltage-clamp protocol allows an estimate of the time constant of inactivation directly (6, 24, 25, 27, 33).


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  		Fig. 6.   Time course of inactivation and recovery from inactivation. A: a 3-stage voltage-clamp protocol (inset) was used to study inactivation of cERG current. First, a cell was voltage clamped at a Vh of -80 mV and then depolarized to a prepotential of +60 mV for 200 ms to activate and partially inactivate cERG current. Next, the cell was briefly repolarized to -100 mV for 2 ms to allow recovery from inactivation without significant deactivation of cERG current and finally depolarized to different voltages to inactivate cERG channels. B: recovery from inactivation was studied using a 2-step protocol (inset). Cells were depolarized for 200 ms to +60 mV from a Vh of -80 mV to activate and inactivate cERG channels and then repolarized to varying voltages to examine the time course of recovery from inactivation. Only current traces during these latter steps to different voltages are shown on an expanded time scale. A single-exponential equation provided the best fit to the cERG current inactivation and recovery from inactivation time courses. C: voltage dependence of the time constants (tau ) for the development of inactivation and recovery from inactivation as a function of membrane potential at 36°C.

The characteristics of cERG inactivation and recovery from inactivation are shown in Fig. 6. cERG current was activated and inactivated by 200-ms depolarizing steps to +60 mV. The cell was then repolarized to -100 mV for 2 ms to allow for recovery from inactivation without significant deactivation of cERG current (27). Finally, a test step was applied to different voltages to measure inactivation of cERG current. The example in Fig. 6A shows that currents elicited by this protocol were large in amplitude and inactivated rapidly. The time constants of inactivation at the different membrane potentials were estimated by fitting a single-exponential function (Eq. 1). These time constants are plotted in Fig. 6C (n = 3-6 cells).

The time constants for recovery from inactivation were measured with a two-pulse protocol (Fig. 6B; see also Refs. 25, 27). A cell was depolarized to +60 mV for 200 ms to activate and inactivate cERG channels; the cell membrane was then repolarized to potentials between -20 and -100 mV. The rapid recovery of cERG current was estimated with a single-exponential fit to the tail current rising phase. The data from multiple cells are summarized and plotted in Fig. 6C. Both the rates of inactivation and the recovery from inactivation were dependent on membrane potential, as seen in Fig. 6C. The time constants of inactivation and recovery ranged between 0.4 and 5 ms depending on membrane potential, and inactivation was much faster than activation of the channel.

Open channel conductance and K+ selectivity. A fully activated current-voltage (I-V) relationship for cERG channels is shown in Fig. 7. cERG current was activated by a depolarizing step to +60 mV for 1 s followed by steps to different membrane potentials, as shown in Fig. 7A. The rationale for this protocol is to open all channels, allowing K+ conductance, and then quickly change the membrane potential to measure the effect of a change in the electrochemical driving force on K+ current through the open channels. At the more positive membrane potentials, cERG current showed apparent inward rectification due to rapid inactivation and the current amplitude was relatively time independent. During steps to more negative membrane potentials, cERG channels rapidly recovered from inactivation to reach a peak value followed by voltage-dependent decay. Figure 7B shows the I-V plot of the peak tail currents observed at the membrane potentials indicated in the abscissa. This relationship demonstrates the apparent inward rectification at membrane potentials positive to -30 mV, with maximum outward current obtained at voltages between -20 and -30 mV. In the standard solutions used in these studies, K+ current recorded at membrane potentials more positive than -85 mV was outward, whereas at more negative membrane potentials the current was inward, defining the Erev under these conditions.


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  		Fig. 7.   Fully activated current-voltage (I-V) relation for cERG channels in HEK-293 cells. A: voltage-clamp protocol and example current traces. Cells were voltage clamped at a Vh of -80 mV and were first depolarized to a prepotential of +60 mV to fully activate and partially inactivate the cERG current and then repolarized to Vtail ranging from -100 to +40 mV in 10-mV increments. B: I-V plot of the peak cERG current vs. Vtail. The maximum current amplitude was at a Vtail of about -30 mV at 36°C (n = 6).

The K+ selectivity of cERG channels was estimated by determining the Erev of the tail current at varying [K+]o. Tail currents were measured with a protocol similar to that in Fig. 7A. Cells were voltage clamped to a test potential of +60 mV for 500 ms followed by repolarizing voltage steps incremented in 5-mV steps between -110 and -45 mV. Erev was determined in each cell as the zero current intercept of the plot of tail current amplitude and was determined at [K+]o of 2, 4, and 10 mM. Erev were -98.5 ± 0.8 (n = 6 cells), -84.2 ± 0.7 (n = 6 cells), and -61.3 ± 0.6 (n = 4 cells) mV at 2, 4, and 10 mM [K+]o, respectively, and when corrected for the 4.9-mV junction potential in the recording system, the measured values were -103, -89, and -66 mV, respectively. The [K+]i was 144 mM. Therefore, the Nernst equation predicts Erev of -111, -93, and -69 mV at 2, 4, and 10 mM [K+]o, respectively, and a slope of 61 mV per 10-fold change in [K+]o for a perfectly K+-selective pore at 36°C.

As [K+]o is lowered, other partially permeant ions such as Na+ influence the measured Erev. This is predicted by the Goldman-Hodgkin-Katz equation (Eq. 4), which provided an excellent fit to these data (not shown). Assuming that K+ and Na+ are the major permeant ions, PNa/PK was 0.007. These data indicate a very high, but not absolute, K+ selectivity for the cERG channel. Although we did not extensively characterize the ionic selectivity of these channels with multi-ion substitutions, our estimate of PNa/PK at physiological concentrations should be a reasonable estimate of K+ selectivity given that Na+ was present at more than 30-fold excess compared with K+.

cERG Pharmacology

Inhibition of cERG K+ current. To explore the pharmacology of cERG K+ channels, we tested the ability of a number of drugs known to inhibit hERG channels, including MK-499, astemizole, terfenadine, and cisapride. These agents represent chemically and structurally diverse compounds from different therapeutic classes. Each has been shown to inhibit hERG channels or IKr in native cardiac myocytes (3, 4, 11, 13, 14, 20, 21, 26, 28, 38). Figure 8 compares inhibition of cERG (Fig. 8A) and hERG (Fig. 8B) by MK-499. Whole cell K+ currents are shown at different membrane potentials in control (no drug) or 30 or 100 nM MK-499. MK-499 potently inhibited both the canine and human channels. Normalized K+ current tails are plotted as a function of membrane potential in Fig. 8, C and D. In each case, K+ current in the presence of MK-499 at the concentration shown was normalized to the predrug control. MK-499 at 30 nM inhibited >50% of the current, and this effect appeared to be independent of membrane potential. Comparison of the inhibition of cERG and hERG channels by MK-499 is shown as concentration-effect curves in Fig. 8E. Data were fit with a Hill equation (Eq. 5) to estimate an IC50 for each condition. The best-fitting IC50 values derived from this type of analysis for MK-499, astemizole, terfenadine, and cisapride are listed in Table 5.


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  		Fig. 8.   Concentration and voltage dependence of MK-499 inhibition of cERG and hERG current. Example current traces for cERG (A) and hERG (B) in control and after superfusion with 30 and 100 nM MK-499 are shown. Voltage-activation relationships were generated from a Vh of -80 mV with 1-s depolarizing steps to Vt ranging from -60 to +50 mV in 10-mV increments, and deactivating tail currents were measured during repolarization to -50 mV at 36°C (inset in A). Data were obtained at apparent steady state (5-8 min) for each concentration of MK-499 at 36°C. Normalized I-V plots of cERG (C) and hERG (D) tail currents during control and after MK-499 at 30 and 100 nM, respectively (n = 4-6) are also shown. E: concentration dependence of cERG and hERG channel inhibition by MK-499. Inhibition of peak repolarizing tail current after a step to a Vt of +40 mV is shown. Peak outward tail currents were measured for each drug concentration and expressed as a fraction of the control value in the same cell. Best-fit IC50 values obtained by nonlinear least-squares regression of the Hill equation (Eq. 5) on individual data are shown (n = 3-7). The solid curve is drawn arbitrarily based on the average IC50.


                              
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  		Table 5.   Comparison of known hERG channel antagonists on cERG vs. hERG channels

Inhibition of [35S]MK-499 binding to cERG channels. The binding site of MK-499 on the hERG channel subunit has been located with alanine scanning mutagenesis and homology modeling (12). Here we compared standard hERG blockers for inhibition of [35S]MK-499 binding to cERG and hERG channels. Figure 9 shows the concentration-dependent displacement of [35S]MK-499 binding by MK-499, astemizole, terfenadine, and cisapride. The apparent affinity of each of these agents is consistent with the degree of inhibition of the expressed current in the voltage-clamp studies (Table 5).


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  		Fig. 9.   Competitive displacement of 35S-labeled MK-499 binding for cERG and hERG channels by MK-499, astemizole, terfenadine, and cisapride. Best-fit IC50 values obtained by nonlinear least-squares regression of the Hill equation (Eq. 5) on individual data are shown (n = 4). The solid curves were drawn arbitrarily based on the average of the fitted IC50 values.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have established HEK-293 cell lines that stably express high levels of functional cERG channels and have characterized their biophysical and pharmacological properties. To our knowledge, this is the first comprehensive evaluation of the properties of cERG in a mammalian expression system at physiological temperature. We also have developed a novel methanesulfonanilide ([35S]MK-499) binding assay and compared displacement of binding with block of K+ current under voltage clamp. We found that cERG channels are kinetically complex, with biophysical properties suitable to control myocyte repolarization. The properties of cERG channels are comparable to those of cardiac IKr (15) and hERG (37).

The gene cerg encodes a protein (cERG) that consists of 1,159 amino acids with 97% identity to hERG (36). Notably, all amino acid differences are outside of the S2-S6 and P domains of the channel protein. Immunoblot analysis revealed two protein bands (155 and 135 kDa) that were each slightly larger than the predicted molecular mass of the core protein (127 kDa) based on nucleotide composition. Zhou et al. (37) reported similar findings for hERG and found that N-glycosidase F treatment of hERG converted both bands to apparent smaller molecular mass, suggesting that both proteins are glycosylated.

Zhou et al. (37) previously established stably transfected HEK-293 cell lines expressing functional hERG channels and characterized their electrophysiological and pharmacological properties. At 35°C, hERG current activated at voltages positive to -50 mV, maximum outward K+ current was reached at +10 mV, and the outward current amplitude decreased at more positive voltages. The hERG channels were highly selective for K+. Voltage-dependent activation of cERG channels occurred between -50 and -10 mV, and the channels were highly K+ selective at 36°C.

Most studies with hERG channels expressed in Xenopus oocytes or mammalian cells have been performed at room temperature (~23°C) (22, 25, 29, 33). Compared with 23°C, cERG current amplitude was increased approximately twofold at 36°C, and the kinetic properties were altered. The greatest temperature effect was on the rate of channel activation. Warming channels from room to body temperature shifted the equilibrium between closed and open states in favor of channel opening by nearly 20 kJ/mol (Delta Delta G = -4.4 kcal/mol). Zhou et al. (37) observed a similar increase in hERG current on heating from 23°C to 35°C. Activation, inactivation, recovery from inactivation, and deactivation kinetics were faster at 36°C than at 23°C and closely resembled those of native IKr at physiological temperatures. These findings characterize the functional properties of cERG channels and emphasize the complexity of the kinetic steps underlying channel gating that is highly temperature dependent.

We can also compare our results with findings reported for native IKr. Studies of the native current are complicated because of its small size and the numerous other ionic currents active in the same range of membrane potentials in native myocytes (15). For these reasons, native IKr usually requires pharmacological separation. A detailed study of human IKr in atrial cells was conducted by Wang et al. (34). In their study, the E-4031-sensitive current activation data at 36°C (pharmacological isolation of IKr) had voltage and time dependence similar to our findings obtained with cERG at 36°C and those of Zhou et al. (37) for hERG. Veldkamp and co-workers (30) studied IKr in human ventricular cells and reported an average activation time constant near 100 ms at +30 mV. In myocytes from some mammalian species studied at physiological temperatures, e.g., guinea pig ventricular cells (23) or canine ventricular cells (9), the time and voltage dependence of IKr activation are also close to what we found with cERG, although in other species such as rabbit ventricular cells (2), slower activation kinetics values have been reported. Apparent differences in kinetics between ERG current and IKr, a concern in several previous reports, are far less at physiological temperatures and may in fact be accounted for by slight variations in experimental conditions including ionic composition of solutions, cell type, and temperature.

Zehelein et al. (36) recently examined, in limited detail, the properties of cERG and hERG expressed in Xenopus oocytes. In that expression system at 20°C, they reported V1/2 values of -36.6 and -30.8 mV for cERG and hERG, respectively. These estimates are substantially more negative than values for cERG and hERG obtained in this study in HEK-293 cells at 23°C and in other studies for hERG expressed in HEK-293 (37) or CHO (37) cells. The value of -30.8 mV is also much more negative than the V1/2 of -15.1 mV originally reported for hERG expressed in Xenopus oocytes (22). The reason for these differences is unclear.

Pharmacological inhibition of IKr in ventricular myocytes or hERG channels leads to the development of aLQT and the potentially lethal polymorphic ventricular arrhythmia torsades de pointes. In this study we have chosen four agents known to prolong QT interval in humans and dogs for pharmacological evaluation on cERG channels. These include a prototypical methanesulfonanilide class III antiarrhythmic (MK-499), two antihistamines (astemizole and terfenadine), and a gastrointestinal prokinetic agent (cisapride). Notably, the latter three prescription drugs have been withdrawn from the U.S. market because of their potential for causing aLQT and sudden death due to arrhythmia.

Previous studies defined the potencies of these agents in various test systems including IKr in guinea pig myocytes and hERG current heterologously expressed in Xenopus oocytes or mammalian cell lines. MK-499 inhibited IKr with an IC50 of 44 nM (Ref. 11), hERG in oocytes with a potency that varied with [K+]o [IC50 = 123 nM (Ref. 27); IC50 = 34 and 120 nM at 2 and 96 mM [K+]o, respectively (Ref. 12)], and hERG in HEK-293 cells with an IC50 of 21 nM in this study. Astemizole potently inhibited IKr [IC50 = 1.5 nM (Ref. 21)], hERG in oocytes [IC50 = 48 nM (Ref. 28)], and hERG in HEK-293 [IC50 congruent  1 nM (Ref. 38 and this study)]. Terfenadine inhibited IKr [IC50 of 50 nM (Ref. 21)], hERG oocytes [IC50 = 350 or 246 nM (Refs. 20, 28)], and hERG in HEK-293 [56 nM (Ref. 18); 9 nM (this study)]. Finally, cisapride inhibited IKr with an IC50 = 15 nM (Ref. 4) and hERG expressed in mammalian cells with potencies ranging from an IC50 of 6.5 to 44.5 nM (Refs. 14, 18, 31 and this study).

In this study, the potency for each of the agents was comparable between hERG and cERG within a given assay, i.e., measurement of expressed current or MK-499 binding (Table 5; Figs. 8 and 9); IC50 values were within approximately twofold. Similarly, the IC50 values between the displacement of MK-499 and inhibition of cERG and hERG currents assays differed by less than fourfold for astemizole, terfenadine, and cisapride, whereas MK-499 was ~30-fold more potent in the MK-499 binding assay. This interesting observation may occur as a consequence of a number of possible factors. First, differences in the assay conditions might have differentially influenced the interaction of MK-499 with ERG channels compared with other agents. These include [K+]o of 4 versus 60 mM, temperature (36° vs. 22°C), intact cells versus isolated membranes, exacting control versus no or limited control of transmembrane potential, and drug exposure times of 5-8 min versus 2 h in the voltage-clamp assay compared with the binding assay, respectively. MK-499 and other methanesulfonanilides, in particular, have been shown to very slowly and preferentially interact with depolarized (open or inactivated) states of the channel (7, 13, 25, 26). The experimental conditions used in the MK-499 binding assay (2 h in 60 mM [K+]o, thereby causing membrane depolarization) are expected to drive the voltage-dependent ERG channels into primarily the open or inactivated state. It has been postulated that ERG channels may undergo both slow and ultraslow (minutes) processes of inactivation (17), and this latter inactivation process would be favored in the binding assay but essentially absent under our voltage-clamp conditions. This could increase the apparent affinity estimate for MK-499 but not for the other non-methanesulfonanilides in the binding assay. Differences in the kinetics of the on-rate for MK-499 compared with the other agents may also influence the ultimate apparent binding affinity estimate expressed as an IC50. In voltage-clamp experiments the drug washin period was of necessity limited to <10 min with application of test pulses at 0.1 Hz. If full equilibration between the drug and the highest affinity state of the channel did not occur under these conditions, then the apparent affinity would be reduced. Nevertheless, there was for the most part good agreement among the assays and the rank order of potency was preserved.

In conclusion, this study defines in detail the functional and pharmacological properties of cERG channels in a mammalian expression system and shows that the canine channel is biophysically and pharmacologically similar to the human channel and the K+ current is similar to native canine IKr. The results suggest that canine ERG is a reasonable surrogate for the human channel. The MK-499 binding displacement assay robustly detected electrophysiologically active compounds and provides a novel tool for detection of drugs affecting ERG channels. Discrepancies in the ability of known inhibitors of hERG to prolong QT in canine models in vivo cannot be best explained by differences at the molecular/channel level but are more likely due to other factors.


    ACKNOWLEDGEMENTS

We thank Dr. Joerg Zehelein for providing the cERG cDNA and Dr. Armando Lagrutta for helpful discussions and suggestions.


    FOOTNOTES

Address for reprint requests and other correspondence: J. J. Salata, Dept. of Molecular Pharmacology, Merck Research Laboratories, WP46-300, 770 Sumneytown Pike, West Point, PA 19486 (E-mail: joseph_salata{at}merck.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 3, 2002;10.1152/ajpheart.00220.2002

Received 13 March 2002; accepted in final form 17 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bennett, PB, and Johnson JP. Molecular physiology of cardiac ion channels. In: Basic Cardiac Electrophysiology and Pharmacology (1st ed.), edited by Zaza A, and Rosen M.. Newark, NJ: Harwood Academic, 2000, p. 29-57.

2.   Clay, JR, Ogbaghebriel A, Paquette T, Sasyniuk BI, and Shrier A. A quantitative description of the E-4031-sensitive repolarization current in rabbit ventricular myocytes. Biophys J 69: 1830-1837, 1995[Web of Science][Medline].

3.   Crumb, WJ, Jr, Wible B, Arnold DJ, Payne JP, and Brown AM. Blockade of multiple human cardiac potassium currents by the antihistamine terfenadine: possible mechanism for terfenadine-associated cardiotoxicity. Mol Pharmacol 47: 181-190, 1995[Abstract].

4.   Drolet, B, Khalifa M, Daleau P, Hamelin BA, and Turgeon J. Block of the rapid component of the delayed rectifier potassium current by the prokinetic agent cisapride underlies drug-related lengthening of the QT interval. Circulation 97: 204-210, 1998[Abstract/Free Full Text].

5.   Johnson, JP, Jr, Balser JR, and Bennett PB. A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels. J Neurosci 21: 4143-4153, 2001[Abstract/Free Full Text].

6.   Johnson, JP, Mullins FM, and Bennett PB. Human ether-a-go-go-related gene K+ channel gating probed with extracellular Ca2+---evidence for two distinct voltage sensors. J Gen Physiol 113: 565-580, 1999[Abstract/Free Full Text].

7.   Kiehn, J, Lacerda A, Wible B, and Brown AM. Molecular physiology and pharmacology of HERG: single channel currents and block by dofetilide. Circulation 94: 2572-2579, 1996[Abstract/Free Full Text].

8.   Limbird, L. Cell Surface Receptors: A Short Course on Theory and Methods. Boston: Nijhof, 1986.

9.   Liu, DW, and Antzelevitch C. Characteristics of the delayed rectifier current (IKr and IKs) in canine ventricular epicardial, midmyocardial, and endocardial myocytes: a weaker IKs contributes to the longer action potential of the M cell. Circ Res 76: 351-365, 1995[Abstract/Free Full Text].

10.   Liu, S, Rasmusson RL, Campbell DL, Wang S, and Strauss HC. Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes. Biophys J 70: 2704-2715, 1996[Medline].

11.   Lynch, JJ, Wallace AA, Stupienski RF, Baskin EP, Beare CM, Appleby SD, Salata JJ, Jurkiewicz NK, Sanguinetti MC, Stein RB, Gehret JR, Kothstein T, Claremon DA, Elliott JM, Butcher JW, Remy DC, and Baldwin JJ. Cardiac electrophysiologic and antiarrhythmic actions of two long-acting spirobenzopyran piperidine class III agents, L-702,958 and L-706,000 [MK-499]. J Pharmacol Exp Ther 269: 541-54, 1994[Abstract/Free Full Text].

12.   Mitcheson, JS, Chen J, Lin M, Culberson C, and Sanguinetti MC. A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci USA 97: 12329-12333, 2000[Abstract/Free Full Text].

13.   Mitcheson, JS, Chen J, and Sanguinetti MC. Trapping of a methanesulfonanilide by closure of the HERG potassium channel activation gate. J Gen Physiol 115: 229-240, 2000[Abstract/Free Full Text].

14.   Mohammad, S, Zhou Z, Gong Q, and January CT. Blockage of the HERG human cardiac K+ channel by the gastrointestinal prokinetic agent cisapride. Am J Physiol Heart Circ Physiol 273: H2534-H2538, 1997[Abstract/Free Full Text].

15.   Nerbonne, JM. Molecular basis of functional voltage-gated K+ channel diversity in the mammalian myocardium. J Physiol 525: 285-298, 2000[Abstract/Free Full Text].

16.   Noble, D, and Tsien RW. Outward membrane currents activated in the plateau range of potentials in cardiac Purkinje fibres. J Physiol 200: 205-231, 1969[Abstract/Free Full Text].

17.   Pennefather, PS, Zhou W, and DeCoursey TE. Idiosyncratic gating of HERG-like K+ channels in microglia. J Gen Physiol 111: 795-805, 1998[Abstract/Free Full Text].

18.   Rampe, D, Roy ML, Dennis A, and Brown AM. A mechanism for the proarrhythmic effects of cisapride (Propulsid): high affinity blockade of the human cardiac potassium channel HERG. FEBS Lett 417: 28-32, 1997[Web of Science][Medline].

19.   Roden, DM, Lazzara R, Rosen M, Schwartz PJ, Towbin J, and Vincent GM. Multiple mechanisms in the long-QT syndrome. Current knowledge, gaps, and future directions. Circulation 94: 1996-2012, 1996[Abstract/Free Full Text].

20.   Roy, M, Dumaine R, and Brown AM. HERG, a primary human ventricular target of the nonsedating antihistamine terfenadine. Circulation 94: 817-823, 1996[Abstract/Free Full Text].

21.   Salata, JJ, Jurkiewicz NK, Wallace AA, Stupienski RF, Guinosso PJ, and Lynch JJ. Cardiac electrophysiological actions of the histamine H1-receptor antagonists astemizole and terfenadine compared with chlorpheniramine and pyrilamine. Circ Res 76: 110-119, 1995[Abstract/Free Full Text].

22.   Sanguinetti, MC, Jiang C, Curran ME, and Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell 81: 299-307, 1995[Web of Science][Medline].

23.   Sanguinetti, MC, and Jurkiewicz NK. Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents. J Gen Physiol 96: 195-215, 1990[Abstract/Free Full Text].

24.   Smith, PL, Baukrowitz T, and Yellen G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379: 833-836, 1996[Web of Science][Medline].

25.   Snyders, DJ, and Chaudhary A. High affinity open channel block by dofetilide of HERG expressed in a human cell line. Mol Pharmacol 49: 949-955, 1996[Abstract].

26.   Spector, PS, Curran ME, Keating MT, and Sanguinetti MC. Class III antiarrhythmic drugs block HERG, a human cardiac delayed rectifier K+ channel. Open-channel block by methanesulfonanilides. Circ Res 78: 499-503, 1996[Abstract/Free Full Text].

27.   Spector, PS, Curran ME, Zou A, Keating MT, and Sanguinetti MC. Fast inactivation causes rectification of the IKr channel. J Gen Physiol 107: 611-619, 1996[Abstract/Free Full Text].

28.   Suessbrich, H, Waldegger S, Lang F, and Busch AE. Blockade of HERG channels expressed in Xenopus oocytes by the histamine receptor antagonists terfenadine and astemizole. FEBS Lett 385: 77-80, 1996[Web of Science][Medline].

29.   Trudeau, M, Warmke JW, Ganetzky B, and Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92-95, 1995[Abstract/Free Full Text].

30.   Veldkamp, MW, van Ginneken ACG, Opthof T, and Bouman LN. Delayed rectifier channels in human ventricular myocytes. Circulation 92: 3497-3504, 1995[Abstract/Free Full Text].

31.   Walker, BD, Singleton CB, Bursill JA, Wyse KR, Valenzuela SM, Qiu MR, Breit SN, and Campbell TJ. Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride: affinity for open and inactivated states. Br J Pharmacol 128: 444-450, 1999[Web of Science][Medline].

32.   Wang, H, Kunkel DD, Martin TM, Schwartzkroin PA, and Tempel BL. Heteromultimeric K+ channels in terminal and juxtaparanodal regions of neurons. Nature 365: 75-79, 1993[Medline].

33.   Wang, S, Liu S, Morales MJ, Strauss HC, and Rasmusson RL. A quantitative analysis of the activation and inactivation kinetics of HERG expressed in Xenopus oocytes. J Physiol 502: 45-60, 1997[Abstract/Free Full Text].

34.   Wang, Z, Fermini B, and Nattel S. Rapid and slow components of delayed rectifier current in human atrial myocytes. Cardiovasc Res 28: 1540-1546, 1994[Web of Science][Medline].

35.   Warmke, JW, and Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA 91: 3438-3442, 1994[Abstract/Free Full Text].

36.   Zehelein, Z, Zhang W, Koenen M, Graf M, Heinemann SH, and Katus HA. Molecular cloning and expression of cERG, the ether a go-go-related gene from canine myocardium. Pflügers Arch 442: 188-191, 2001[Web of Science][Medline].

37.   Zhou, ZF, Gong QM, Ye B, Fan Z, Makielski JC, Robertson GA, and January CT. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J 74: 230-241, 1998[Medline].

38.   Zhou, ZF, Vorperian VR, Gong QM, Zhang ST, and January CT. Block of HERG potassium channels by the antihistamine astemizole and its metabolites desmethylastemizole and norastemizole. J Cardiovasc Electrophysiol 10: 836-843, 1999[Web of Science][Medline].

Am J Physiol Heart Circ Physiol 284(1):H256-H267
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