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(Circulation Research. 2001;89:33.)
© 2001 American Heart Association, Inc.

Cellular Biology

Molecular Interactions Between Two Long-QT Syndrome Gene Products, HERG and KCNE2, Rationalized by In Vitro and In Silico Analysis

Reza Mazhari, Joseph L. Greenstein, Raimond L. Winslow, Eduardo Marbán, H. Bradley Nuss

From the Department of Medicine (R.M., E.M., H.B.N.), Whitaker Biomedical Engineering Institute and Center for Computational Medicine and Biology (J.L.G., R.L.W., E.M.), and Institute of Molecular Cardiobiology (R.M., R.L.W., E.M., H.B.N.), Johns Hopkins University, Baltimore, Md.

Correspondence to H. Bradley Nuss, PhD, Institute of Molecular Cardiobiology, Johns Hopkins School of Medicine, 720 Rutland Ave, Ross Building 844, Baltimore, MD 21205. E-mail bradnuss{at}mail.jhmi.edu

Abstract

Abstract—The cardiac delayed rectifier potassium current mediates repolarization of the action potential and underlies the QT interval of the ECG. Mutations in either of the two molecular components of the rapid delayed rectifier (I_K,r), HERG and KCNE2, have been linked to heritable or acquired long-QT syndrome. Mechanisms whereby mutations of KCNE2 produce fatal cardiac arrhythmias characteristic of long-QT syndrome remain unclear. In this study, we characterize functional interactions between HERG and KCNE2 with a view to defining underlying mechanisms for action potential prolongation and long-QT syndrome. Whereas coexpression of hKCNE2 with HERG alters both kinetics and density of ionic current, incorporation of these effects into a quantitative model of the action potential predicts that only changes in current density significantly affect repolarization. Thus, the primary functional consequence of hKCNE2 on action potential morphology is through modulation of I_K,r density, as predicted by the model. Mutations associated with long-QT syndrome that result only in modest changes of gating kinetics may be epiphenomena or may modulate action potential repolarization via interaction with alternative pore-forming potassium channel subunits.

Key Words: delayed rectifier potassium channels • Markov chains • action potential • arrhythmia • accessory proteins

It has been shown that human ether-à-go-go–related gene (HERG)^{1 2} encodes the pore-forming subunit of the rapid delayed rectifier potassium channel (I_K,r).^{1 3} Abbott et al⁴ recently showed that channels formed by coexpression of KCNE2 (encoding minK-related peptide 1, MiRP1) and the pore-forming subunit HERG resemble native cardiac I_K,r channels more closely in their gating and unitary conductance, modulation by extracellular potassium, and inhibition by class III antiarrhythmic medications (eg, E-4031).⁴ More importantly, they identified mutations in hKCNE2 (eg, Q9E and M54T) that were associated with acquired long-QT syndrome and ventricular fibrillation. Various mutations in HERG have also been linked with the familial form of long-QT syndrome.^{5 6} Therefore, whereas lesions in either of the two molecular components of I_K,r, HERG and hKCNE2, have been linked to heritable or acquired long-QT syndrome, mechanisms whereby genetic lesions of hKCNE2 produce fatal cardiac arrhythmias remain unclear. Mutant hKCNE2, when coexpressed with HERG, can result in alterations of both current density and kinetics or of kinetics alone.⁴ This raises the question of whether altered current density or channel kinetics contributes more significantly to action potential prolongation and arrhythmia in patients with these genetic lesions. Better understanding of functional interactions between these two gene products could facilitate development of superior therapeutic approaches for particular lesions in either HERG or hKCNE2.

Our first objective, therefore, is to characterize the functional effects of hKCNE2 coexpression with HERG. Because ion channel gating models provide a quantitative description of gating behavior and give clues to channel structure,⁷ the second objective is to develop a Markov state model of both HERG and HERG-hKCNE2 coassembly (Figure 1) and use this model to additionally elucidate mechanisms of interactions. Because mutations in both HERG and hKCNE2 have been identified to be associated with long-QT syndrome,^{5 6} a third objective is to predict the consequences of HERG-hKCNE2 interactions for action potential repolarization by incorporating the newly developed Markov model into a mathematical model of the cardiac action potential.⁸

View larger version (10K): [in this window] [in a new window]   Figure 1. State diagram of the HERG and HERG+hKCNE2 Markov model. Cl, C2, and C3 are closed states, O is the open state, and I is the inactivated state. All s and ßs are a function of voltage (ie, =A · exp[BVm] and ß=C · exp[DVm], where Vm is the membrane potential). Kf and Kb are voltage independent.12 is defined as a function of other transition rates to satisfy the microscopic reversibility condition.

Materials and Methods

Transfection of HEK 293 Cells
Stably transfected, G-418–resistant HEK 293 cell lines coexpressing HERG channels and green fluorescent protein (GFP) were used for the experiments.⁹ . These cells were transiently transfected with either 0.8 µg of hMiRP1 cDNA (hKCNE2) (kindly provided by Dr Steve Goldstein, Yale University, New Haven, Conn) and 0.2 µg of red fluorescent protein (RFP) (DsRed, Clontech) or 1 µg of RFP alone using Lipofectamine (Gibco BRL). GFP- and RFP-positive cells were studied within 24 to 36 hours of transfection.

Patch-Clamp Recording
Membrane currents were measured using whole-cell patch clamp.¹⁰ Cells were bathed in solution containing (in mmol/L) NaCl 140, KCl 5.4, MgCl₂ 1, HEPES 10, glucose 10, and CaCl₂ 2, pH 7.4 (adjusted with NaOH). Borosilicate glass pipettes were pulled and fire polished to final tip resistances of 5 to 6 M when filled with pipette solution containing (in mmol/L) KCl 140, MgCl₂ 1, HEPES 10, EGTA 5, and MgATP 4, pH 7.3 (adjusted with KOH). Uncompensated capacitance currents in response to small hyperpolarizing voltage steps were recorded for offline integration as a means of measuring cell capacitance. All recordings were obtained at room temperature.

Voltage protocols used in this study are as follows, with holding potential of -80 mV in all cases. (1) Voltage-dependent activation: prepulse for 3.5 seconds from -80 to 60 mV in 10-mV steps, test pulse to -50 mV for 0.5 seconds. (2) Activation kinetics: incremental prepulse duration from 0.035 to 0.49 seconds at 0 to 60 mV in 20-mV steps, test pulse for 0.2 seconds at -120 mV. (3) Deactivation kinetics and current-voltage relation: prepulse for 2 seconds at 60 mV, test pulse at -140 to 40 mV in 10-mV steps for 1 second. (4) Inactivation kinetics, steady-state inactivation, and instantaneous current-voltage relation: prepulse to 60 mV for 0.5 seconds, pulse to -80 for 30 ms, test pulse at -80 to 60 mV in 10-mV steps for 0.2 seconds. (5) Recovery from inactivation: prepulse to 60 mV for 0.3 seconds, test pulse at -100 to 20 mV in 10-mV steps for 0.2 seconds.

Mathematical Model
The structure of the Markov model is shown in Figure 1. This model is based on previous work of Liu et al¹¹ and Wang et al¹² for I_K,r and HERG currents, respectively. Closed-state inactivation (C₃I) (Figure 1) was added to the scheme to accommodate recent results of Kiehn et al¹³ on single-channel studies on HERG, which showed that rapid inactivation may take place from the closed states. A model parameter set yielding optimal fit to experimental data was obtained using the Neldar-Mead Simplex method (Matlab, MathWorks) as described previously.¹⁴

A model of the canine cardiac midmyocardial action potential was used.⁸ The existing I_K,r component of the model was replaced with the newly developed Markov representation of HERG or HERG-hKCNE2. The rates in both models were adjusted for temperature to match kinetics of I_K,r at 37°C using experimental reports of Zhou et al¹⁵ (using Q₁₀ of 3.3). Temperature effects were assumed to be similar for both HERG and HERG-hKCNE2 channels. Channel conductance (G_Kr) was set to 0.0203 mS/µF to match the reported I_K,r current densities reported by Li et al¹⁶ for human cardiac myocytes, which are similar to those reported in canine. Therefore, the baseline or wild-type I_K,r model is based on HERG-hKCNE2 kinetics (Table) and G_Kr conductance, and comparisons are made with this set of parameters (eg, Figure 6). The mutation model of HERG-hKCNE2 (M54T) is based on the data reported by Abbott et al.⁴ The parameters in the HERG-hKCNE2 model were adjusted to closely represent the reported currents and place in the action potential model to evaluate the effects of the mutation on action potential duration. The tenth action potential in response to each cycle length stimulus is reported here to ensure that model responses have reached a steady state. (The action potential models are available for download from http://perspolis.bme.jhu.edu.)

View this table: [in this window] [in a new window]   Table 1. HERG and HERG-hKCNE2 Markov State Transition Rates

View larger version (17K): [in this window] [in a new window]   Figure 6. Effects of alterations in kinetics or conductivity on action potential repolarization. A, Effects of increase in conductivity alone (GKr2GKr). B, Effect of changes in kinetics (K) alone (from HERG-hKCNE2 to HERG model, Table). C, Effects of changes in conductivity (GKr2GKr) and kinetics (K). D, Effects of M54T-hKCNE2 mutation on action potential repolarization. Lower panels in C and D represent IK,r currents corresponding to each case in these perturbations during action potential. Wild-type (HERG-hKCNE2 kinetics and GKr conductance) action potentials and IK,r currents are shown in thick black lines. E, Changes in APD90 relative to wild-type models (APD90) as function of cycle length for each case described in above panels (symbols match the symbols shown above each action potential trace). Negative and positive values indicate a decrease and an increase in APD90 compared with wild-type model, respectively. Cycle length of 1 second in panels A through D (arrow in panel E). Horizontal scale bar=0.1 second; vertical scale bar=0.2 pA/pF.

Statistical Analysis
All the results referred to as HERG are RFP-only transfection of the stable cells (n=5), and the HERG+hKCNE2' group is RFP and hKCNE2 cotransfection (n=6). All data shown are mean±SEM. ANOVA was used for statistical analysis, and P<0.05 was considered statistically significant. Solid lines in all figures represent the Markov model results unless otherwise specified.

Results

We tested whether coassembly with hKCNE2 alters activation properties of HERG and found that addition of hKCNE2 did not affect voltage-dependent activation. There was no significant difference in the half-maximal activation voltage (-9.5±5.2 versus -8.6±3.4 mV for HERG and HERG-hKCNE2, respectively) determined by fitting the experimental data to a Boltzmann function; there was also no significant difference in the slope factors (7.2±0.3 versus 7.4±0.4 mV for HERG and HERG-hKCNE2, respectively) (Figure 2A). Addition of hKCNE2, however, resulted in a change of activation kinetics (Figure 2B). Compared with HERG alone, coassembly with hKCNE2 resulted in an acceleration of activation with the largest effect at the lowest potential (0 mV) (Figure 2C, protocol 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Activation characteristics of HERG and HERG-hKCNE2 coassembly. A, Voltage-dependent activation (using protocol 1, normalized peak tail currents). B, Activation kinetics (using protocol 2) at 0 (triangles), 20 (circles), 40 (diamonds), and 60 mV (squares). C, Activation time constants () as a function of voltage. Filled symbols indicate HERG; open symbols, HERG-hKCNE2; and solid lines, model results. *P<0.05.

Coassembly of HERG and hKCNE2 also affects deactivation kinetics (Figure 3B). Analysis of tail currents (Figure 3A, tail currents of protocol 3) shows that deactivation time constants () are decreased on average by 40% (P<0.0001). At potentials positive to the reversal potential (-82 mV), there is no significant difference in deactivation rate constants. For example, at -70 mV, deactivation time constants were 331±44 and 475±83 ms for HERG and HERG-hKCNE2, respectively. Addition of hKCNE2 also affects kinetics of inactivation (Figure 4B). Coassembly of hKCNE2 and HERG results in significant reduction of inactivation time constants at all potentials (P<0.0001, using protocols 4 and 5). There is a 68% reduction in inactivation time constants for potentials 0 mV and a 39% reduction for potentials >0 mV (Figure 4B). However, there is no significant change in the voltage dependence of steady-state inactivation (half-maximal inactivation voltage of -34.6±5.6 versus -38.9±3.2 mV and slope factor of 20.1±2.2 versus 20.3±0.9 mV for HERG versus HERG-hKCNE2, respectively) (Figure 4A). Therefore, coassembly of hKCNE2 and HERG affects only the rates of development and recovery from, but not the voltage dependence of, inactivation.

View larger version (15K): [in this window] [in a new window]   Figure 3. Deactivation characteristics. A, Representative normalized experimental (top) and model (bottom) tail currents (protocol 3). B, Deactivation time constants () (using tail currents in protocol 3, as shown in panel A). Filled symbols indicate HERG; open symbols, HERG-hKCNE2; and solid lines, model results.

View larger version (18K): [in this window] [in a new window]   Figure 4. Inactivation characteristics of HERG and HERG-hKCNE2. A, Voltage-dependent steady-state inactivation (using tail currents in protocol 4). B, Inactivation time constants () (using tail currents in protocols 4 and 5). Filled symbols indicate HERG; open symbols, HERG-hKCNE2; and solid lines, model results.

Coassembly of hKCNE2 and HERG results in a significant decrease of inward (40%) and outward (55%) current density (Figure 5A, P<0.001; using peak tail currents of protocol 3), as confirmed by a 2-fold reduction (P<0.001) in the slope of the instantaneous current-voltage relationship (from 2.9±0.5 to 1.5±0.3 pS/pF, P<0.05, using peak tail currents of protocol 4) (Figure 5B).

View larger version (20K): [in this window] [in a new window]   Figure 5. Current-voltage relations. A, Current densities (I) as a function of voltage (peak tail currents, protocol 3, normalized to membrane capacity). B, Instantaneous current-voltage relation (using peak-tail currents of protocol 4). Filled symbols indicate HERG; open symbols, HERG-hKCNE2; and solid lines, linear fits to the data.

Rate constants associated with the Markov models (Figure 1) representing HERG and HERG-hKCNE2 coexpression are shown in the Table. Consistent with our experimental data, with the exception of _o and _i (see Figure 1), all rate constant variation resulting from coassembly is voltage independent (ie, no change in B of A · exp[BV_m]). Moreover, inactivation from the closed state (C3I) is nearly voltage independent and has magnitude significantly smaller than the open to inactivated state transition rate (OI). Although addition of hKCNE2 accelerates closed-state inactivation, this rate is still significantly smaller than that of open-state inactivation. The rate of recovery from inactivation to the closed state (IC3) is significantly smaller than recovery to the open state (IO), with largest values at the most hyperpolarized potentials (ie, V_m<-80 mV). In summary, addition of hKCNE2 to HERG only alters kinetics of channel gating, with no change in voltage dependence.

Whereas the results shown thus far shed light on the functional consequences of coexpression of hKCNE2 and HERG, they do not provide an explanation as to how deletion of hKCNE2 from the channel structure, or a mutation associated with it, would affect cardiac action potential repolarization and ultimately lead to long QT in the affected patients. To address this issue, we have simulated effects of the observed changes associated with this coassembly on action potential repolarization using a computational model of the action potential⁸ at various pacing cycle lengths (Figure 6E). Although addition of hKCNE2 to HERG produces considerable changes in the kinetics of channel gating, these changes have little effect on action potential repolarization (Figure 6B). Replacing rate constants from the HERG-hKCNE2 model to the HERG model (Table) results in an 18-ms reduction in action potential duration measured at 90% repolarization (APD₉₀) (Figure 6B). Inclusion of changes in both kinetics and conductance (increasing G_Kr 2-fold) results in a 62-ms reduction in APD₉₀ (Figure 6C), whereas an increase in conductance alone produces a 40-ms reduction in APD₉₀ (Figure 6A). It is clear from these data that presence of a loss-of-function mutation would result in a decrease in action potential duration as a result of an increase in peak I_K,r density in phase 3 of the action potential (Figures 6C and 6D, bottom panels). These changes were magnified at shorter cycle lengths (Figure 6E), indicating that perhaps alterations observed here may figure prominently at higher heart rates (eg, during ß-adrenergic stimulation or exercise). Thus, whereas coexpression of hKCNE2 with HERG alters both the kinetics and density of ionic current, incorporation of these data into a quantitative model of the action potential predicts that changes in current density exert the greatest effect on repolarization.

We have also used the HERG-hKCNE2 model to predict effects of a point mutation associated with hKCNE2 (M54T).⁴ As reported previously, this mutation results in a 2- to 3-fold increase in deactivation rates and a modest reduction in activation slope factor compared with HERG-WT hKCNE2 coexpression.⁴ Here rate constants of the HERG-hKCNE2 Markov model (Table) were adjusted on the basis of data presented by Abbott et al⁴ to include kinetic changes associated with M54T-hKCNE2 mutation and incorporated in the action potential model (Figure 6D). These rate changes result in an increase in APD₉₀ by 30 ms. More importantly, the prolongation of action potential was magnified at shorter cycle lengths (Figure 6E; eg, a 50-ms prolongation in APD₉₀ at 500-ms cycle length compared with a 30-ms prolongation at 1000-ms cycle length). This finding helps to rationalize previous clinical observations in which M54T-hKCNE2 mutation resulted in prolongation of the QT interval in the ECG of the affected patient during exercise.⁴

Discussion

In this study we took an integrative approach with a view to defining the underlying mechanisms by which HERG and hKCNE2 interact and the way in which this interaction affects action potential repolarization. We first characterized the functional effects of hKCNE2 coexpression with HERG and found that addition of hKCNE2 to HERG accelerates the kinetics of activation, deactivation, and inactivation, with no change in the voltage dependence of activation and inactivation. Coassembly also results in a significant reduction in current density compared with channels formed by HERG alone. Whereas the alterations observed in the kinetics and current densities confirm those in the original report on HERG-KCNE2 coassembly,⁴ we did not observe any change in the voltage dependence of activation. This discrepancy may be attributable to the differences in the expression system used (oocyte expression system as opposed to HEK293 cells used here) and in the voltage protocols applied.

Because ion channel gating models provide a quantitative description of gating behavior and give clues to channel structure-function relationships,⁷ a Markov state model of both HERG and HERG-hKCNE2 coassembly (Figure 1) was developed and used to additionally elucidate mechanisms of interactions. Consistent with the experimental observations, optimization of parameters in these models suggested that addition of hKCNE2 to HERG only alters kinetics of channel gating, with no change in voltage dependence (Table). Because mutations in both HERG and hKCNE2 have been associated with long-QT syndrome,^{5 6} we predicted the consequences of HERG-hKCNE2 interactions for action potential repolarization by incorporating the newly developed Markov model into a mathematical model of the cardiac action potential.⁸ Whereas changes in both the kinetics and the density of ionic current alter action potential repolarization, modeling predicts that changes in current density exert the greatest effect on repolarization, and these effects are more pronounced at shorter cycle lengths (ie, at higher heart rates). The latter may have a significant relevance to mutations associated with hKCNE2 that result in a prolongation of action potential (as we have illustrated here for the case of M54T-hKCNE2). Note that this result is in contrast to what is seen in LQT3 patients (SCN5A mutations, attributable to persistent inward sodium current),^{17 18} where action potential prolongation and early afterdepolarization are observed during longer cycle lengths (ie, lower heart rates), as opposed to shorter cycle lengths observed here.

In summary, our principal finding is that kinetic changes associated with coexpression of hKCNE2 and HERG have little effect whereas changes of channel conductance have large effects on action potential repolarization. This finding rationalizes the pathogenicity of clinical mutations, which results in a modest alteration of kinetics and larger changes of current densities, in which the affected patients are susceptible to arrhythmias and long-QT syndrome (eg, M54T-hKCNE2 and I57T-hKCNE2).⁴ These conclusions can be generalized to include other genes in the KCNE family (eg, S74L-KCNE1 and D76N-KCNE1).^{6 19 20} Furthermore, if there are mutations identified in hKCNE2 that result in a modest alteration in kinetics alone (eg, recent work of Schulze-Bahr et al²¹ on hKCNE2) and yet patients show susceptibility to arrhythmias, it is conceivable that these phenotypes are a result of additional, not yet fully understood interactions between these peptides (hMiRP1) and an alternative potassium subunit (eg, KCNQ1²² ). This is consistent with the present expanded view of protein function where each protein in the cell membrane functions as part of an extended web of interacting molecules.²³ The integrative and iterative approach of combining gene expression, functional analyses, and mathematical modeling enables us to demonstrate underlying functional mechanisms not evident from each individual component alone.

Acknowledgments

This work was supported by the American Heart Association Scientist Development Grant and NIH/NHLBI R01 HL66381 to H.B.N. R.M. was supported by NIH postdoctoral training grant HLO7227. R.L.W. was supported by NIH/NHLBI R01 HL60133, the Whitaker Foundation, and the Falk Medical Trust. General laboratory support was provided by NIH/NHLBI Specialized Center of Research grant P50 HL52307. E.M. holds the Michel Mirowski, M.D. Professorship of Cardiology of the Johns Hopkins University.

Footnotes

Original received February 23, 2001; revision received May 29, 2001; accepted May 29, 2001.

This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

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