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Cardiovascular Research 2006 71(4):695-703; doi:10.1016/j.cardiores.2006.06.017
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Copyright © 2006, European Society of Cardiology

Functional modulation of the transient outward current Ito by KCNE β-subunits and regional distribution in human non-failing and failing hearts

Susanne Radickea, Diego Cotellaa, Eva Maria Grafa, Ulrich Bansea, Norbert Jostb, András Varróc, Gea-Ny Tsengd, Ursula Ravensa and Erich Wettwera,*

aMedical Faculty, Dresden University of Technology, Fetscherstr. 74, Dresden, 01307 Germany
bDivision of Cardiovascular Pharmacology, Hungarian Academy of Sciences, 6701 Hungary
cDepartment of Pharmacology & Pharmacotherapy, Faculty of Medicine, University of Szeged, Dom ter 12, Szeged, 6701 Hungary
dVirginia Commonwealth University, Department of Physiology-Box 980551, Richmond, Virginia 23298-055, USA

* Corresponding author. Department of Pharmacology and Toxicology, Medical Faculty, Dresden University of Technology, Fetscherstr. 74, Dresden, 01307 Germany. Tel.: +49 351 458 6278; fax: 49 351 458 6315. Email address: erich.wettwer{at}tu-dresden.de

Received 10 March 2006; revised 2 June 2006; accepted 13 June 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives The function of Kv4.3 (KCND3) channels, which underlie the transient outward current Ito in human heart, can be modulated by several accessory subunits such as KChIP2 and KCNE1–KCNE5. Here we aimed to determine the regional expression of Kv4.3, KChIP2, and KCNE mRNAs in non-failing and failing human hearts and to investigate the functional consequences of subunit coexpression in heterologous expression systems.

Methods We quantified mRNA levels for two Kv4.3 isoforms, Kv4.3-S and Kv4.3-L, and for KChIP2 as well as KCNE1–KCNE5 with real-time RT-PCR. We also studied the effects of KCNEs on Kv4.3+KChIP2 current characteristics in CHO cells with the whole-cell voltage-clamp method.

Results: In non-failing hearts, low expression was found for KCNE1, KCNE3, and KCNE5, three times higher expression for KCNE2, and 60 times higher for KCNE4. Transmural gradients were detected only for KChIP2 in left and right ventricles. Compared to non-failing tissue, failing hearts showed higher expression of Kv4.3-L and KCNE1 and lower of Kv4.3-S, KChIP2, KCNE4, and KCNE5. In CHO cells, Kv4.3+KChIP2 currents were differentially modified by co-expressed KCNEs: time constants of inactivation were shorter with KCNE1 and KCNE3-5 while time-to-peak was decreased, and V0.5 of steady-state inactivation was shifted to more negative potentials by all KCNE subunits. Importantly, KCNE2 induced a unique and prominent 'overshoot' of peak current during recovery from inactivation similar to that described for human Ito while other KCNE subunits induced little (KCNE4,5) or no overshoot.

Conclusions: All KCNEs are expressed in the human heart at the transcript level. Compared to Ito in native human myocytes, none of the combination of KChIP2 and KCNE produced an ideal congruency in current characteristics, suggesting that additional factors contribute to the regulation of the native Ito channel.

KEYWORDS Gene expression; Heart failure; Ventricular function; K-channel; Membrane currents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The transient outward current (Ito) determines the early repolarization phase of cardiac action potential and thus contributes to the determination of the repolarization phase as well as excitation–contraction coupling via modulating Ca2+ and other K+ currents [1,24]. In human ventricle Kv4.3 (KCND3) is the main pore-forming {alpha}-subunit of Ito [11], and short and long splice variants (Kv4.3-S and Kv4.3-L) were detected [10,14]. The electrophysiological properties of Ito are modulated by several β-subunits [9] among them KChIP2 (K+-channel interacting protein) is the most thoroughly investigated. KChIP2 increases peak Ito current density by promoting trafficking of Kv4.3 to the cell membrane [2], slows inactivation, and accelerates recovery from inactivation [8,23].

KCNE1 (minK) is the major accessory subunit of the KvLQT1 (KCNQ1) channel forming the slow delayed rectifier current IKs [5]. KCNE2 encoding for MinK-related peptide 1 (MiRP1) is also expressed in human myocardium [12] and associates with the cardiac Kv4.3 protein [1]. Kv4.3 has been shown to be modulated by KCNE1 and KCNE2 in heterologous systems [9]. However, the kinetic properties of native Ito in human ventricular myocytes can not be properly explained by Kv4.3 interaction with KChIP2, KCNE2 or KCNE1 [9]. Other accessory β-subunits with accelerating effects on the Ito current kinetics may contribute to the characteristics of Ito. One potential candidate is the dipeptidyl-aminopeptidase-like protein (DPP6), which has been recently identified in neuronal and heart tissue and can substantially accelerate the inactivation of transient K+ currents [19,21]. Furthermore, KCNE3, KCNE4 and KCNE5 exhibit pronounced and distinct effects on important potassium channel β-subunits such as KCNQ1 and HERG [3,17]. It is conceivable that these promiscuous KCNE proteins also interact with Kv4.3 and influence expression and kinetics of Ito.

Native Ito exhibits a characteristic transmural gradient in ventricles, with larger Ito density in epicardium than in endocardium [18,27]. In human and dog hearts, this Ito gradient is believed to be determined by differential KChIP2 expression [22], although there is no consensus on this point [8]. In failing myocardium Ito is reduced in epicardial as well as endocardial tissue layers [13,18,27], and this is correlated with a reduction of Kv4.3 mRNA [13]. Little is known about the influence of heart failure on the expression of KChIP2, or other putative Ito β-subunits.

The aim of the present investigation was to quantify the mRNA levels of Ito {alpha}-subunit isoforms, Kv4.3-L and Kv4.3-S, and putative accessory β-subunits KCNE1–KCNE5 and KChIP2 in human non-failing and failing ventricles using the real-time RT-PCR technique. Our main finding is that the expression of {alpha}- and β-subunits was differentially regulated in failing hearts. In functional expression experiments in CHO cells we found distinct patterns of modulation of Kv4.3+KChIP2 gating kinetics by KCNE subunits, which suggest that in addition to Kv4.3+KChIP2 association with KCNE2 and other still not identified subunits or regulators are required to mimic the native Ito function in human heart.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Tissue
Tissue samples were collected from explanted hearts of NYHA IV patients (four male, one female) with DCM with written consents. Medication included digitoxin, metoprolol and torasemide. Healthy tissue was derived from five donor hearts (two male, three female). Biopsies were excised from the central region of the anterior wall of left (LV) and right (RV) ventricles, were separated into subepi-(epicardial) and subendocardial (endocardial) layers and immediately stored in liquid nitrogen. The study conformed with the Declaration of Helsinki.

2.2. Molecular biology
Total RNA was isolated using the LiCl-method [25]. Quantitative real-time RT-PCR was performed as described in detail recently [21]. Primer pairs specific for human Kv4.3-L and Kv4.3-S, KChIP2 and KCNE1–KCNE4 were intron-spanning. KCNE5 primers were modified from published sequences [16] (Table 1).


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  		Table 1 Primers and conditions for PCR

 
2.3. CHO expression system
Chinese hamster ovary (CHO) cell lines stably transfected with hKv4.3-L or hKv4.3/hKChIP2 were donated from Sanofi–Aventis (Frankfurt, Germany) and cultured as described in Ref. [21]. KCNE1-3 and KCNE5 cDNAs were obtained from human ventricle mRNA and cloned in pIRES2-EGFP (BD Biosciences, Heidelberg, Germany). pXOOM-KCNE4 was donated from Dr. M. Grunnet (Copenhagen, Denmark). Both pIRES and pXOOM plasmids drive cDNA expression under the control of CMV promoters and use EGFP as a transfection marker. KCNE1-5 plasmids were transfected using 2.5 µl of Roti-Fect® transfection reagent (Carl Roth, Karlsruhe, Germany) and a total amount of 0.5 µg plasmid each.

2.4. Electrophysiological experiments
Whole-cell patch-clamp experiments were performed using a HEKA-EPC8 amplifier (HEKA Elektronik, Lambrecht, Germany). Bath solution contained (in mM): NaCl 150, KCl 5.4, MgCl2 2, CaCl2 1.8, HEPES 10, glucose 11, pH 7.4. Pipette solution contained (in mM): KCl 40, potassium aspartate 80, NaCl 8, CaCl2 2, MgATP 5, EGTA 5, GTP 0.1, HEPES 10, pH 7.4 adjusted with KOH. Pipette tip resistance was between 2.5 and 3.5 M{Omega} when filled with pipette solution.

The holding potential was – 80 mV. Currents were measured with clamp steps between – 60 and +60 mV. Series resistance was compensated up to 85%. Clamp pulse generation, data collection and analysis were performed with ISO2 software (MFK, Niedernhausen, Germany). Data were not corrected for junction potentials which was calculated with 11 mV for the electrode solution with JPCalc software (P.H. Barry, Sydney, Australia). All experiments were performed at room temperature (22 °C).

2.5. Statistics
Results are given as means±S.E.M. Statistical analysis was performed with Student's t-test or one-way ANOVA with Bonferroni's post hoc test (GraphPad Prism software, V4.1; San Diego, CA). Differences were considered to be significant if P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Differential remodeling of Kv4.3 isoforms and KChIP2 expression in human failing heart
The two isoforms, Kv4.3-S and Kv4.3-L, were homogeneously expressed with similar mRNA levels in all regions of non-failing hearts. In failing heart, their expression was differentially regulated. While Kv4.3-L was upregulated, Kv4.3-S was markedly downregulated, so that the long isoform clearly dominated (Fig. 1A, Table 2). KChIP2 expression showed a steep transmural gradient in non-failing hearts in both left and right ventricles. In failing hearts KChIP2 expression was markedly reduced in epicardial tissue of both ventricles. The reduction in endocardial layers of both left and right ventricles was less, although differences between pooled data were significant. The transmural gradient of KChIP2 was preserved in failing hearts although to a reduced level (Fig. 1A, 2A, Table 2).


Figure 1
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  		Fig. 1 mRNA expression of Kv4.3, KChIP2 and KCNE in human non-failing (NF) and failing hearts (HF). (A) Kv4.3-L, Kv4.3-S and KChIP2 mRNA expression. (B) KCNE1–KCNE5 mRNA. Mean values±S.E.M. of mRNA ([fg] of total isolated RNA [ng]) (*P<0.05, **P<0.01, ***P<0.001), n=5.

 

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  		Table 2 Summary of quantitative mRNA expression data in all samples from 5 non-failing and 5 failing hearts

 
3.2. Expression pattern and remodeling of KCNE1–KCNE5 in heart failure
In specimens from non-failing hearts the mRNA expression of KCNE subunits was substantially smaller by a factor of 5 to 10 compared to the {alpha}-subunit Kv4.3 and KChIP2, with the exception of KCNE4, the expression of which was 6 times higher than for Kv4.3 (Fig. 1, Table 2). The order of expression level was KCNE4>>Kv4.3-L~Kv4.3-S>KCNE2>KCNE1~KCNE3~KCNE5. All KCNE β-subunits, with the exception of KCNE2, were homogeneously expressed between the two ventricles of non-failing human hearts. For KCNE2, we detected a significantly larger expression in epicardial specimen of the right compared to the left ventricle (Fig. 2B, Table 2). Unlike expression of KChIP2, KCNE2 did not exhibit an expression gradient between epi-and endocardium in right ventricle. In left ventricle KCNE2 expression tended to be smaller in epicardial than endocardial tissue thus exhibiting a transmural gradient opposite in direction to that of KChIP2.


Figure 2
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  		Fig. 2 Regional expression of KChIP2 and KCNE2 mRNA in human heart. KChIP2 and KCNE2 mRNA was determined in subendocardial (endo) and subepicardial (epi) layers of RV and LV in non-failing (NF) and failing (HF) hearts (n=5). (A) KChIP2 expression in epi and endo layers of RV and LV in NF and HF. (B) KCNE2 mRNA in epi and endo of RV and LV in NF and HF. Mean values±S.E.M.

 
In samples from failing hearts the expression levels of KCNEs were differentially altered. While expression of KCNE1 was significantly larger, that of KCNE4 and KCNE5 was lower compared to non-failing hearts. For KCNE2 and KCNE3, the mRNA levels were not different in non-failing and failing hearts (Fig. 1B, Table 2).

3.3. Effects of KCNE1–KCNE5 on Kv4.3+KChIP2 stably expressed in CHO cells
3.3.1. Current density and current-voltage relations
None of the 5 KCNE subunits was able to produce membrane currents when co-expressed with Kv4.3-L alone (Fig. 3A). Only in combination with KChIP2, expression of Kv4.3-L led to prominent transient outward currents (Fig. 3 B). KChIP2 is an obligatory accessory subunit of native Ito channels in the heart [15]. Therefore, Kv4.3-L+KChIP2 stably expressed in CHO cells served as control, and KCNE subunits were transiently co-expressed to test their effects on the Ito kinetics. Kv4.3-L+KChIP2 had an average peak outward current density of 303±54 pA/pF (+50 mV, n=34). In the presence of KCNE subunits, current densities were not significantly altered (Table 3).


Figure 3
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  		Fig. 3 Effects of KChIP2 and KCNE4 on Kv4.3 currents. Current tracings (initial 300 ms) elicited by test steps (1000 ms) from – 80 mV to – 40, – 20, 0,+20,+40 and +60 mV in CHO cells stably expressing (A) Kv4.3-L+KCNE4, (B) Kv4.3-L+KChIP2 (C) Kv4.3-L+KChIP2+KCNE4, (D) normalized currents at +50 mV to compare rate of inactivation.

 

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  		Table 3 Electrophysiological parameters of Ito at room temperature in CHO cells and human ventricular myocytes

 
3.3.2. Time course of activation and inactivation
All KCNE subunits accelerated the time course of activation, as indicated by the reduction in time-to-peak current by about 50% (Fig. 4A). The smallest effect was detected with the combined expression of KCNE2 plus KCNE4. The time course of Ito inactivation, which could be best described by a two-exponential function, was also affected by KCNEs. KCNE3, KCNE4 and KCNE5 significantly reduced the fast time constant of inactivation. KCNE4 and KCNE5 also markedly reduced the slow time constant of inactivation (data not shown). Therefore, co-expression of KCNE4 and KCNE5 resulted in a pronounced acceleration of the inactivation time-course (shown for KCNE4 in Fig. 3 C,D; mean data in Fig. 4C, Table 3).


Figure 4
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  		Fig. 4 Effects of KCNE1–KCNE5 (E1–E5) on Kv4.3+KChIP2 current activation and inactivation. (A) Time course of activation is characterized by time to reach peak current (ms). Inset: tracings of current activation for Kv4.3+KChip2 and Kv4.3+KChip2+KCNE4 at +50 mV. (B) Normalized steady-state activation (gm) curves for Ito calculated from I/V-curves assuming an Erev of – 60 mV. (C) Fast time constant of inactivation determined from fitting biexponential function to individual current traces at +50 mV; (D) Normalized steady-state inactivation (I/Imax) curves for Ito. In panels (B) and (D), mean data were fitted by Boltzmann functions to estimate half maximum voltage (V0.5, dotted lines). Compare Table 3.

 
3.3.3. Voltage-dependence of activation and inactivation
The activation curves were calculated from current-voltage relations assuming a constant reversal potential of – 60 mV [18]. The voltage-dependence of Ito activation was not markedly altered by co-expression of KCNEs (Fig. 4B, Table 3). KCNE β-subunits had, however, a more pronounced impact on the voltage-dependence of Ito current inactivation. All KCNE β-subunits shifted V0.5 of steady-state inactivation to more negative membrane potentials (Fig. 4D, Table 3).

3.3.4. Recovery from inactivation
The time course of recovery from inactivation was determined with a double-pulse protocol starting at – 80 mV to +50 mV including a recovery interval at – 80 mV of 5 to 5000 ms. KCNE co-expression had differential impacts on the kinetics of recovery from inactivation. KCNE1, KCNE2, KCNE3 and KCNE2 plus KCNE4 slowed the time course of recovery. KCNE4 alone, however, accelerated recovery from inactivation (Fig. 5, Table 3). In addition KCNE2, KCNE4 and KCNE5 produced a so-called "overshoot" in Ito current amplitude, a phenomenon described for native Ito in human ventricular myocytes [27]. The "overshoot" implies that with recovery intervals up to 1000 ms peak amplitude of Ito was transiently larger than during the reference pulse, with a maximum at the recovery interval of 200 ms. The amplitude of the "overshoot" was largest with KCNE2 and amounted to about 10% for the reference amplitude. Control cells (Kv4.3+KChIP2) and cells co-expressing KCNE1 and KCNE3 did not manifest an "overshoot" during recovery from inactivation (Fig. 5, Table 3).


Figure 5
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  		Fig. 5 Effects of KCNEs on the time course of Kv4.3+KChIP2 current recovery from inactivation. (A) Time course of recovery from inactivation of Ito, test pulse +50 mV, recovery potential – 80 mV. Curve fitting to a biexponential function [Y=A*(1 – exp(–  t/{tau}fast))+B* exp(–  t/{tau}slow)+C] yielded {tau}fast-values of 53±7 ms for Kv4.3+KChIP2 (n=26), 89±13 ms for Kv4.3-L+KChIP2+KCNE3 (n=13), 38±4 ms for Kv4.3-L+KChIP2+KCNE4 (n=11). Expanded time scale to focus on fast time course of recovery. (B) Same data as in (A) compressed time scale focussing on "overshoot" phenomenon. Complete data in Table 3. (C) Original current traces of Kv4.3-L+KChIP2 and (D) Kv4.3-L+KChIP2+KCNE2 during recovery from inactivation.

 
3.3.5. Co-expression of the two KCNE subunits KCNE2 plus KCNE4
Since co-expression of individual KCNE subunits produced different current characteristics of Ito, we tested whether a combined expression of KCNE2 (inducing the largest Ito overshoot) and KCNE4 (having the largest expression among KCNE subunits) led to a better recapitulation of native Ito kinetics. With KCNE2 plus KCNE4 the overshoot was preserved, V0.5 of current inactivation, however, was rather positive and time constant of recovery was slowed. Thus, the effects of KCNE2 dominated those of KCNE4.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
4.1 Kv4.3 and KChIP2 as two major components of Ito channels in human heart
In human heart, Kv4.3 is the major ion conducting {alpha}-subunit underlying the transient outward current. Previous studies reported a long (Kv4.3-L) and a short isoform (Kv4.3-S) [15], whose electrophysiological characteristics are identical [10]. Kv4.3-L possesses 2 additional PKC phosphorylation sites and represents the isoform which is susceptible to PKC-dependent reduction in current amplitude via {alpha}-adrenoceptor stimulation [20].

In the present study we separately examined the expression levels of Kv4.3-L and Kv4.3-S in failing and non-failing hearts, but since we did not have access to a plasmid for Kv4.3-S, functional aspects of the channel in the expression system could only be investigated with Kv4.3-L. This approach can be justified if the identity of Kv4.3-L and Kv4.3-S with respect to the proposed binding site for KChIP2 at the N-terminal domain is taken into account. The 2 isoforms also exhibit homology in their transmembrane and extracellular domains where KCNE2 and other KCNE subunits are supposed to bind (Tseng, unpublished observations). Therefore, the interactions with KChIP2 and KCNE subunits are likely to be similar for both Kv4.3 isoforms.

In non-failing hearts, Kv4.3-L and Kv4.3-S are expressed in similar mRNA quantities with no transmural gradient. In failing hearts, Kv4.3-L is up-regulated and Kv4.3-S is down-regulated, leading to a predominant expression of Kv4.3-L. Nevertheless, the sum of Kv4.3-L and Kv4.3-S is reduced in failing compared to non-failing hearts, which confirms the well known heart failure-associated down-regulation of global Kv4.3 and Ito amplitude [13]. Furthermore, high plasma concentrations of noradrenaline in chronic heart failure can stimulate cardiac {alpha}-adrenoceptors and activate PKC. The predominant expression of Kv4.3-L which is sensitive to PKC phosphorylation (see above) may contribute to reduced Ito function.

KChIP2 is required for proper trafficking of Kv4.3 channels to the cell membrane [2,4,15]. In various expression systems, i.e. Xenopus oocytes and HEK-293 cells, co-expression of Kv4.3 with KChIP2 enhances Ito current density, slows current inactivation and accelerates recovery from inactivation [8,28]. We could not study an effect of KChIP2 on Ito kinetics because unlike the other expression systems, CHO cells did not exhibit Ito when stably transfected with Kv4.3 only. In any case, co-expression of Kv4.3+KChIP2 in CHO cells yields current with a time course of inactivation similar to native cardiac Ito, but markedly slower recovery from inactivation and steady-state inactivation in a more positive potential range. These findings suggest that KChIP2 may not be the only accessory subunit of native Ito in cardiomyocytes [12], although we cannot exclude that other regulatory processes may also be involved.

We did not distinguish between various KChIP2 isoforms [7], because there is no consensus as to which ones are present in the heart. Nevertheless, we confirm the differential epi-/endocardial expression of KChIP2, underlying the steep transmural gradient in Ito [22,23]. In heart failure, KChIP2 is substantially down-regulated which can further contribute to the reduced amplitude of Ito [13].

4.2 Role of KCNE subunits in native Ito?
In heterologous expression systems, several putative accessory proteins such as KCNEs, DPP6, KChAPs, Kvβ, and even the sodium channel β-subunit NaChβ1 [9] have been found to modulate the properties of Kv4.3 channels. Here we have focused on the KCNE-protein family and all KCNE subunits were detected at the mRNA level in human heart. At the protein level, only KCNE2 has been demonstrated in human heart [12].

Depending on the expression system, there have been conflicting reports as to whether and how KCNE1 can modulate Kv4.3 or the related Kv4.2 channels. In oocytes, KCNE1 did not affect Kv4.2 gating kinetics [28]. In HEK293 cells KCNE1 slowed all kinetic parameters of Kv4.3 and increased current amplitude [9]. A similar regulatory role of KCNE1 in human hearts seems unlikely, since KCNE1 up-regulation in heart failure is accompanied by a reduction in Ito amplitude instead of the expected increase. Kinetic parameters of Ito in CHO cells co-expressing the standard combination of Kv4.3+KChIP2 with KCNE1 reveal better congruency with native Ito for time course of inactivation and steady-state inactivation but recovery from inactivation is slowed and an "overshoot" is absent.

KCNE2 is a promiscuous β-subunit and can interact with several K+ channels (HERG, KCNQ1, KCNQ4, Kv3.4) in heterologous expression systems [17]. When co-expressed with Kv4.3 in Xenopus oocytes in the absence of KChIP2, KCNE2 significantly slows the time course of Ito activation and inactivation and shifts the voltage dependence of activation to positive membrane potentials [28]. However, in our CHO cell expression system KCNE2 appears to have opposite effects. We found that KCNE2 is exceptional because its co-expression can best reproduce the unique "overshoot" phenomenon during recovery from inactivation of human left ventricular epicardial Ito [27], while other KCNE subunits were either ineffective or induced only a small "overshoot" (Table 3). Therefore we suggest that KCNE2 could be an important component of the native Ito channel complex at least in human epicardial cardiomyocytes [28].

Expression level of KCNE3 in cardiac tissue is low and may therefore not have a major effect on Ito kinetics in vivo. KCNE3 induced, however, the largest shift in Ito steady-state inactivation to negative potentials close to the value reported for native myocytes. All other kinetic parameters were less similar to native Ito compared to Kv4.3+KChIP2 alone. In addition, KCNE3 did not abolish outward rectification of Ito as described for KCNQ1 [26].

KCNE4 is the most abundant of all KCNE subunits and even more abundant than the {alpha}-subunit Kv4.3 in human hearts. The former finding confirms recently published data [6,16] although the absolute amount of mRNA for KCNE4 was lower than in our study. This apparent discrepancy is probably due to different primers and general PCR conditions which preclude direct comparison of quantitative expression data from different groups. Because of its exceptionally high expression level KCNE4 is likely to regulate the native Ito. In our experiments KCNE4 accelerated Kv4.3+KChIP2 current inactivation kinetics, shifted voltage dependence of steady-state inactivation to more negative membrane potentials and accelerated recovery from inactivation. Therefore, the properties of the heterologous Ito with KCNE4 as an additional subunit more closely resemble native Ito.

Expression of KCNE5 is also low and we suggest that KCNE5 does not have a prominent role in Ito kinetics. Nevertheless, KCNE5 also yields a rather narrow fit to the kinetics of native Ito including a small "overshoot" in recovery from inactivation.

When co-expressing both KCNE2 and KCNE4 with Kv4.3+KChIP2, the overshoot was still preserved and inactivation of Ito remained accelerated. On the other hand, the time course of inactivation is slowed and steady-state inactivation is shifted to positive values. Therefore, even the combination of the 2 KCNE subunits with the standard channel did not provide a perfect match with native Ito.

In failing hearts, the expression of KCNE subunits was differentially regulated: Expression of KCNE1 was roughly doubled, that of KCNE2 and KCNE3 remained unchanged, and expression of KCNE4 and KCNE5 was reduced. However, despite the failure-associated reduction in absolute mRNA level, KCNE4 remained the largest of the KCNE subunits in failing hearts.

4.3. Study limitations
Tissues samples originated from a heterogeneous patient group. In addition tissue from failing hearts was exposed to chronic drug therapy. Therefore it cannot be excluded that part of the differences originate from drug exposure or are based on differences in the disease state. mRNA content though determined quantitatively does not necessarily correlate with the respective protein levels. Since protein expression has been reported only for KCNE2, it is not known whether the native KCNE protein levels are sufficiently high to have an impact on Ito regulation.

The attempt to identify the composition of the native channel complex by co-expression of putative {alpha}- and β-subunits can only provide incomplete information because neither the stoichiometry of expressed subunits nor the effectiveness of the interaction can be controlled. The similarity in current characteristics compared to native currents is an indirect indication of subunit interaction and has to be confirmed by other means such as immunoprecipitation or fluorescence resonance energy transfer between tagged subunits.

4.4. Conclusion
In the failing heart the changes in the mRNA expression levels of the {alpha}-subunit of Ito and its putative accessory β-subunits show substantial up-and down-regulation. Assuming that these changes are translated into membrane protein expression they may contribute to alterations in cardiac electrophysiology and to risk for arrhythmias of the diseased heart. KCNEs do not substitute for KChIP2 in promoting channel trafficking. The heterologous co-expression of KCNE proteins in CHO cell support the functional interaction of KCNE β-subunits with Kv4.3 in native myocytes, with KCNE2 and possibly KCNE4 being the likely candidates.


   Acknowledgements
 
This work was supported by the MeDDrive 2001 of the Medical Faculty, Dresden University of Technology, Dresden, Germany and by the European Commission, Marie Curie Development Host Fellowship, Contract No.: HPMD-CT-2001-00119. We would like to thank Mrs. Fischer and Mrs. Schöne for their excellent technical assistance. We acknowledge the great help and cooperation of the surgeons of the Dresden Heart Centre (Drs. M. Knaut, K. Matschke, R. Cichon, U. Kappert, M. Tugtekin). We also want to thank Dr. T. Christ for his indispensable assistance and helpful discussion.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Abbott G.W., Goldstein A.N. Potassium channel subunits encoded by the KCNE gene family: physiology and pathophysiology of the MinK-related peptides (MiRPs). Mol Int (2001) 1:95–107.
  2. An W.F., Bowlby M.R., Betty M., Cao J., Ling H.P., Mendoza G., et al. Modulation of A-type potassium channels by a family of calcium sensors. Nature (2000) 403:553–556.[CrossRef][Medline]
  3. Angelo K., Jespersen T., Grunnet M., Nielsen M.S., Klaerke D.A., Olesen S.P. KCNE5 induces time-and voltage-dependent modulation to the KCNQ1 current. Biophys J (2002) 83:1997–2006.[Abstract/Free Full Text]
  4. Bahring R., Dannenberg J., Peters H.C., Leicher T., Pongs O., Isbrandt D. Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating. J Biol Chem (2001) 276:23888–23894.[Abstract/Free Full Text]
  5. Barhanin J., Lesage F., Guillemare E., Fink M., Lazdunski M., Romey G. KvLQT1 and IsK (minK) proteins associate to form the IKs cardiac potassium current. Nature (1996) 384:78–80.[CrossRef][Medline]
  6. Bendahhou S., Marionneau C., Haurogne K., Larroque M.-.M., Derand R., Szuts V., et al. In vitro molecular interactions and distribution of KCNE family with KCNQ1 in human heart. Cardiovasc Res (2005) 67:529–538.[Abstract/Free Full Text]
  7. Decher N., Barth A.S., Gonzalez T., Steinmeyer K., Sanguinetti M.C. Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents. J Physiol (2004) 557:761–772.[Abstract/Free Full Text]
  8. Deschenes I., DiSilvestre D., Juang G.J., Wu R.C., An W.F., Tomaselli G.F. Regulation of Kv4.3 current by KChIP2 splice variants: a component of native cardiac I(to)? Circulation (2002) 106:423–429.[Abstract/Free Full Text]
  9. Deschenes I., Tomaselli G.F. Modulation of Kv4.3 current by accessory subunits. FEBS Lett (2002) 528:183–188.[CrossRef][ISI][Medline]
  10. Dilks D., Ling H.P., Cockett M., Sokol P., Numann R. Cloning and expression of the human Kv4.3 potassium channel. J Neurophysiol (1999) 81:1974–1977.[Abstract/Free Full Text]
  11. Dixon J.E., Shi W., Wang H.S., McDonald C., Yu H., Wymore R.S., et al. Role of the Kv4.3 K+ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ Res (1996) 78:659–668.
  12. Jiang M., Zhang M., Tang D.G., Clemo H.F., Liu J., Holwitt D., et al. KCNE2 protein is expressed in ventricles of different species and changes in its expression contribute to electrical remodeling in diseased hearts. Circulation (2004) 109:1783–1788.[Abstract/Free Full Text]
  13. Käab S., Dixon J., Duc J. Molecular basis of transient outward potassium current down-regulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation (1998) 98:1383–1393.[Abstract/Free Full Text]
  14. Kong W., Po S., Yamagishi T., Ashen M.D., Stetten G., Tomaselli G.F. Isolation and characterization of the human gene encoding Ito: further diversity by alternative mRNA splicing. Am J Physiol (1998) 275:H1963–H1970.[ISI][Medline]
  15. Kuo H.C., Cheng C.F., Clark R.B., Lin J.J., Lin J.L., Hoshijima M., et al. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia. Cell (2001) 107:801–813.[CrossRef][ISI][Medline]
  16. Lundquist A.L., Manderfield L.J., Vanoye C.G., Rogers C.S., Donahue B.S., Chang P.A., et al. Expression of multiple KCNE genes in human heart may enable variable modulation of IKs. J Mol Cell Cardiol (2005) 38:277–287.[CrossRef][Medline]
  17. McCrossan Z.A., Abbott G.W. The MinK-related peptides. Neuropharmacology (2004) 47:787–821.[CrossRef][ISI][Medline]
  18. Nabauer M., Beuckelmann D.J., Uberfuhr P., Steinbeck G. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of human left ventricle. Circulation (1996) 93:168–177.[Abstract/Free Full Text]
  19. Nadal M.S., Ozaita A., Amarillo Y., Vega-Saenz de Miera E., Ma Y., Mo W., et al. The CD26-Related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron (2003) 37:449–461.[CrossRef][ISI][Medline]
  20. Po S.S., Wu R.C., Juang G.J., Kong W., Tomaselli G.F. Mechanism of alpha-adrenergic regulation of expressed hKv4.3 currents. Am J Physiol (2001) 281:H2518–H2527.[ISI]
  21. Radicke S., Cotella D., Graf E.M., Ravens U., Wettwer E. Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative β–subunit of human cardiac transient outward current encoded by Kv4.3. J Physiol (2005) 565:751–756.[Abstract/Free Full Text]
  22. Rosati B., Pan Z., Lypen S., Wang H.S., Cohen I., Dixon J.E., et al. Regulation of KChIP2 potassium channel beta subunit gene expression underlies the gradient of transient outward current in canine and human ventricle. J. Physiol (2001) 533:119–125.[Abstract/Free Full Text]
  23. Rosati B., Grau F., Rodriguez S., Li H., Nerbonne J.M., McKinnon D. Concordant expression of KChIP2 mRNA, protein and transient outward current throughout the canine ventricle. J Physiol (2003) 548:815–822.[Abstract/Free Full Text]
  24. Sah R., Ramirez R.J., Oudit G.Y., Gidrewicz D., Trivieri M.G., Zobel C., et al. Regulation of cardiac excitation–contraction coupling by action potential repolarization : role of the transient outward potassium current (Ito). J Physiol (2003) 546:5–18.[Abstract/Free Full Text]
  25. Sambrook J., Russel D.W. Molecular cloning: a laboratory manual. (2001) vol. 1. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
  26. Schroeder B.C., Waldegger S., Fehr S., Bleich M., Warth R., Greger R., et al. A constitutively open potassium channel formed by KCNQ1 and KCNE3. Nature (2000) 403:196–199.[CrossRef][Medline]
  27. Wettwer E., Amos G.J., Posival H., Ravens U. Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin. Circ Res (1994) 75:473–482.[Abstract/Free Full Text]
  28. Zhang M., Jiang M., Tseng G.N. MinK-related peptide 1 associates with Kv4.2 and modulates its gating function: potential role as beta subunit of cardiac transient outward channel? Circ Res (2001) 88:1012–1019.[Abstract/Free Full Text]

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