Andersen mutations of KCNJ2 suppress the native inward rectifier current I_K1 in a dominant-negative fashion

Abstract

Objective: The Andersen's syndrome is a hereditary disease, which is characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Recently, mutations of the KCNJ2 gene, which encodes the inward rectifying potassium channel subunit Kir2.1, have been identified in affected individuals. However, the functional effects of these mutations have not yet been fully elucidated. Methods and Results: To clarify this situation we generated known Andersen disease mutants of KCNJ2 which did not yield any measurable K⁺ currents in CHO cells indicating that the Andersen mutants failed to form functional homomultimeric complexes. EGFP-tagged KCNJ2 wild-type and mutant channels distributed in a similar homogeneous pattern in the cell membrane suggesting that protein trafficking was not altered by the Andersen mutations but rather implicating that the mutations rendered the KCNJ2 channel non-functional. In heterologous coexpression experiments the Andersen mutants exerted a dominant-negative effect on wild-type KCNJ2. However, the extent of suppression varied between the different KCNJ2 mutants. Given our results in CHO cells, we expressed the disease mutant KCNJ2-S136F in neonate rat cardiomyocytes using adenoviral gene transfer to test the effect of Andersen mutants on native I_K1. I_K1 density was indeed significantly reduced in KCNJ2-S136F-infected cells (n = 9) compared to control cells (n = 9) over a voltage range from −70 to −150 mV (P<0.05). Conclusion: These results support that Kir2.x channels are a critical component of native I_K1 in neonate rat cardiomyocytes and that a dominant-negative suppression of I_K1 in native cells is the pathophysiological correlate of the Andersen's syndrome.

KCNJ2 channels, Inward rectifier, Adenovirus, Gene transfer, Electrophysiology

Time for primary review 25 days.

1 Introduction

The cardiac action potential is based on a tight balance of depolarizing and repolarizing ion currents. In many cell types, including cardiac muscle cells, inward rectifier channels play an important role in setting the resting membrane potential and modulating excitability [1]. The potassium channels with the most pronounced inward rectification belong to the Kir2.x subfamily, which consists of the four members Kir2.1–Kir2.4. Expression of the three inward rectifier channel subunits Kir2.1, Kir2.2 and Kir2.3 has been demonstrated in human heart [2,3]. Similar to voltage-gated potassium channels, Kir subunits are believed to form tetrameric complexes [4]. Transient coexpression experiments indicate that members of the Kir2 subfamily can heteromultimerize [5]. In the heart Kir2 channels are particularly important because they largely determine the shape of the terminal phase of the cardiac action potential [6]. During the plateau of the action potential, inward rectifier channels are mostly closed. However, near the resting potential during the late phase of repolarization and during diastole, the inward rectifier channels provide the dominant membrane conductance. Thus, reduction of the inward rectifier current is expected to increase the propensity for arrhythmias.

Andersen's syndrome is a rare disorder characterized by periodic paralysis, cardiac arrhythmias, and craniofacial dysmorphic features [7,8]. Andersen's syndrome occurs sporadically or is inherited in an autosomal dominant fashion. Recently, mutations in the potassium channel gene KCNJ2, which encodes the Kir2.1 subunit, have been identified in affected individuals [9–11]. However, penetrance is extremely variable, with some mutant carriers displaying little or no phenotype. In the present study, we elucidated the effect of known Andersen mutations on KCNJ2 channel function and protein trafficking. Our results indicate that the mutant channels are non-functional and suppress wild-type channels in a dominant-negative manner, whereas protein trafficking of the mutant channel proteins seems to be unaffected. The observed variable penetrance might in part be due to differences in the amount of the dominant-negative effect. Secondly, using adenoviral gene transfer we could demonstrate that the Andersen disease mutation KCNJ2S136F indeed suppresses native I_K1 in cardiomyocytes.

2 Methods

2.1 Plasmid construction and adenovirus preparation

The adenovirus shuttle vectors pAdEGI, pAdCGI, pAdC-DBEcR, and the expression plasmid pCGI-Kv1.3AYA and pCGI-KCNJ2 (Kir2.1) have been described [12–14]. The full-length coding sequence of hKCNJ2 was cloned into the multiple cloning site of pAdCGI, to generate pAdCGI-KCNJ2. The known human Andersen mutations D71V, del95-98, S136F, G144S, R218W, G300V, E303K and del314/315 were introduced into hKCNJ2 by site-directed mutagenesis, creating the vectors pAdCGI-KCNJ2D71V, pAdCGI-KCNJ2del95-98, pAdCGI-KCNJ2S136F, pAdCGI-KCNJ2G144S, pAdCGI-KCNJ2R218W, pAdCGI-KCNJ2G300V, pAdCGI-KCNJ2E303K and pAdCGI-KCNJ2del314/315, respectively [9]. Fusion constructs of the enhanced green fluorescent protein (EGFP) tagged to the N-terminus of wild-type (pADCGKCNJ2) and mutant KCNJ2 were generated by deletion of the EGFP stop codon and the internal ribosome entry site. The full-length coding sequence of hKCNJ2S136F was cloned into the multiple cloning site of pAdEGI, to give pAdEGI-KCNJ2S136F. Adenovirus vectors were generated as previously described [13–16].

2.2 Transient transfections

Twenty-four hours prior to transfection, CHO-K1 cells (ATCC CCL 61, American Type Culture Collection, Manassas, VA, USA) were seeded at a density of 2.0×10⁵ per 35 mm. Cells were transfected with 0.5 μg/well plasmid DNA of wild-type channels and/or 1 μg/well plasmid DNA of mutant channels (as indicated) using Lipofectamine Plus (Life Technologies, Gaithersburg, MD, USA) as directed by the manufacturer. After 4 h, transfection media were replaced with normal growth media.

2.3 Myocyte isolation and adenovirus infection

A standard trypsin dissociation method was used to prepare ventricular myocytes of 1–2-day-old neonatal rats [14,17]. For voltage-clamp experiments, 3- to 5-day-old monolayer cultures were dispersed by trypsin, and re-plated at a low density to study isolated cells within 2 to 8 h. Infection of neonatal cells was performed 1 to 3 days after plating at a multiplicity of infection (MOI) of 15 to 100 p.f.u. per cell. Cells were incubated for 4 h at 37°C, after which the infection medium was replaced with culture medium. Expression was induced by addition of ponasterone A 10 μM (Invitrogen, San Diego, CA, USA) for 36–60 h.

2.4 Electrophysiology

Experiments were carried out using standard microelectrode whole-cell patch clamp techniques [12,18] with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA, USA) while sampling at 10 kHz and filtering at 2 kHz. Current recordings were performed at room temperature (21 to 23°C). The recording bath solution contained (mmol/l) NaCl 135, KCl 5, CaCl₂ 2, glucose 10, MgCl₂ 1, HEPES 10; pH was adjusted to 7.4 with NaOH. For I_K1 recordings of cardiomyocytes, CdCl₂ 200 μmol/l, and 4-aminopyridine 4 mmol/l were added to block I_CaL, and I_to, respectively. The micropipette electrode solution was composed of (mmol/l): K-glutamate 130, KCl 15, NaCl 5, MgCl₂ 1, HEPES 10, and Mg-ATP 5; pH was adjusted to 7.3 with KOH. Borosilicate microelectrodes had tip resistances of 2–4 MΩ when filled with the internal recording solution. Data were not corrected for the liquid junction potential of −18 mV.

Instantaneous I_K1/I_Kir2.1 current size was measured 10 ms after hyperpolarization to voltages as indicated. A xenon arc lamp was used to view EGFP at 488/530 nm (excitation/emission). Pooled data are presented as mean±standard error of the mean (S.E.M.). Comparisons between groups were performed using one-way ANOVA. P-values less than 0.05 were deemed significant.

2.5 Confocal imaging

Images were taken on a confocal laser scanning microscope (Leica Microsystems, Heidelberg, Germany) with a 40× oil immersion objective lens. Confocal images represent a single scan through one confocal plane. GFP was imaged with an argon laser at 488/520±15 nm (excitation/emission).

3 Results

3.1 Andersen mutants fail to form functional homomultimeric channels

Various mutant K⁺ channels have been reported to form non-functional channels or channels with altered current kinetics [13,19]. To assess the effect of Andersen disease mutants, CHO cells were transfected with wild-type or mutant KCNJ2. All vectors were bicystronic also expressing the enhanced green fluorescent protein (EGFP) under control of a single CMV promoter for easy identification of transfected cells. Overexpression of KCNJ2 resulted in a nearly instantaneous K⁺ current consistent with previous reports of heterologous expression of this ion channel subunit (Fig. 1A) [3,20]. Mean current density at −100 mV was 21.4±1.5 pA/pF (n = 17) (Fig. 1D). In contrast, expression of KCNJ2-D71V, -del95-98, -S136F, -G144S, -R218W, -G300V, -E303K and -del314/315 did not yield any measurable K⁺ currents. Representative original current recordings of KCNJ2-R218W and KCNJ2-E303K, and mean current densities are depicted in Fig. 1B–D. This indicated that these Andersen mutants failed to form functional homomultimeric channels. This might have been due to a loss of function of mutant KCNJ2 subunits, to an alteration of intracellular protein trafficking of functionally intact channel subunits or to a combination of both.

Fig. 1

KCNJ2 mutants fail to form functional homomultimeric channels when heterologously expressed in CHO cells. Overexpression of wild-type KCNJ2 resulted in an instantaneous inwardly rectifying current upon de-/hyperpolarization to voltages of 50 to −100 mV (A). Conversely, original current recordings of KCNJ2-R218W (B) and KCNJ2-E303K (C), and mean current densities measured at −100 mV (D) indicate that expression of Andersen mutants did not yield any measurable current.

3.2 Distribution patterns of KCNJ2 mutants are similar to KCNJ2 wild-type

To clarify whether a loss of function and/or an alteration of protein trafficking is the predominant effect of the various Andersen mutations we generated fusion constructs of the enhanced green fluorescent protein (EGFP) tagged to the N-terminus of KCNJ2 wild-type and mutant channels. Current recordings of the wild-type fusion protein in CHO cells indicated that EGFP did not alter Kir2.1 channel expression or function. Using confocal imaging we evaluated protein distribution patterns of EGFP-tagged wild-type and mutant KCNJ2. As clearly shown in Fig. 2A, expression of EGFP-tagged wild-type KCNJ2 resulted in a homogeneous localization in the cell membrane. Series of fluorescence confocal scanning revealed that each EGFP-tagged mutant was distributed in a similar homogeneous pattern in the cell membrane as wild-type channels. Representative images of EGFP-tagged KCNJ2-R218W and KCNJ2-E303K are depicted in Fig. 2B and C, respectively. This indicated that protein trafficking appeared not to be altered by the Andersen mutations but rather implicated that the mutations rendered the KCNJ2 channel non-functional.

Fig. 2

Distribution patterns of EGFP-tagged KCNJ2 mutant channels are similar to KCNJ2 wild-type. Confocal laser images obtained by argon laser scanning of KCNJ2 wild-type (A), KCNJ2-R218W (B) and KCNJ2-E303K (C) EGFP-fusion constructs show homogeneous localization in the cell membrane.

3.3 Andersen mutants suppress Kir2.1 currents in a dominant-negative manner when coexpressed with wild-type KCNJ2

Several non-functional, mutant K⁺ channels have been demonstrated to exert a dominant-negative effect when coassembling with wild-type subunits [12,13]. To test whether the Andersen mutations result in dominant-negative ion channel constructs, CHO cells were cotransfected with wild-type KCNJ2 and KCNJ2 mutants, or with wild-type KCNJ2 and a non-functional unrelated Kv1.3 construct carrying a pore mutation (Kv1.3-AYA, to exclude any unspecific effects of the KCNJ2 mutants). Representative current recordings of KCNJ2 wild-type coexpressed with KCNJ2-R218W and KCNJ2-E303K (Fig. 3B and C), and mean current densities (Fig. 3D) demonstrate that I_Kir2.1 was significantly suppressed by all tested KCNJ2 mutants (−100 mV, P<0.05) confirming dominant-negative properties of the Andersen mutations. However, the extent of suppression varied between the different KCNJ2 mutants. The strongest dominant-negative effect was observed for KCNJ2-S136F (3.99±0.55 pA/pF, n = 6) and KCNJ2-R218W (4.11±0.70 pA/pF, n = 7), the weakest effect for KCNJ2-G144S (13.45±2.02 pA/pF, n = 5) compared to the wild-type control (21.42±1.57 pA/pF, n = 17).

Fig. 3

Effect of KCNJ2 mutants on wild-type KCNJ2 heterologously expressed in CHO cells. Representative current recordings of KCNJ2-R218W (B) and KCNJ2-E303K (C), and mean current densities measured at −100 mV (D) show that KCNJ2 Andersen mutants significantly decreased I_Kir2.1 in a dominant-negative manner when the mutants were coexpressed with wild-type KCNJ2 channels.

3.4 KCNJ2-S136F suppresses native I_K1 in neonatal cardiomyocytes

Kir2.x is thought to underlie the major part of the native inward rectifier current I_K1 in cardiomyocytes. Given our observations in CHO cells, overexpression of a construct carrying KCNJ2-S136F in neonate rat cardiomyocytes should reduce or eliminate native I_K1 if Kir2.x channels are indeed the major molecular components of this cardiac current, and if Andersen mutants also exhibit dominant-negative effects in native cells. Therefore, I_K1 current recordings were performed in neonatal control ventriculocytes and in myocytes expressing KCNJ2-S136F. For increased expression efficiency, viral gene transfer techniques were employed to deliver our genes of interest using bicystronic adenoviral vectors, which express the KCNJ2 gene and EGFP under the control of an ecdysone inducible promoter [21]. Ecdysone responsiveness was conferred by coinfection with the receptor virus AdC-DBEcR and stimulation with the ecdysone analog ponasterone A [12,14]. I_K1 density was indeed markedly reduced in KCNJ2-S136F-infected cells (n = 9) compared to control cells (n = 9) (Fig. 4A–C). I_K1 suppression by KCNJ2-S136F reached statistical significance over a voltage range of −70 to −150 mV (P<0.05). These results confirm that Kir2.x channels are a critical component of native I_K1 in neonate rat cardiomyocytes and that the Andersen mutation KCNJ2-S136F also exerts a dominant-negative effect in native cells.

Fig. 4

The Andersen mutant KCNJ2-S136F suppressed native I_K1 in neonate cardiomyocytes. Representative original current traces of native I_K1 recorded in a non-infected neonatal myocyte (A) and in a KCNJ2-S136F-infected cell (B), and mean current densities (C) demonstrate the dominant-negative effect of KCNJ2-S136F on native I_K1. I_K1 suppression by KCNJ2-S136F reached statistical significance between −70 to −150 mV (* indicates P<0.05).

4 Discussion

Heterologous expression of mutant ion channel subunits and transgene expression in native cells have been useful tools to dissect the mechanisms and contributions of various disease mutations and wild-type ion channel genes to excitability and arrhythmogenesis [12,13,16]. In the present study we employed these strategies to confirm for the first time that Andersen mutants suppress native cardiac I_K1 in a dominant-negative manner. Furthermore, our results clearly indicate that Kir2.x channels are the major molecular component of native I_K1 in neonate rat ventriculocytes, and that Andersen mutations undermine KCNJ2 channel function but do not alter protein trafficking.

The Andersen's syndrome is a heterogeneous disorder characterized by periodic paralysis of skeletal muscles and dysmorphic features. Additionally, most affected patients demonstrate cardiac manifestations which include a prolongation of the QT interval and ventricular arrhythmias [7,8]. Recently, in some of these patients mutations in the KCNJ2 potassium channel gene which encodes the Kir2.1 subunit have been identified [9–11]. This led to the proposal that Andersen's syndrome should be considered as subtype of the long QT syndrome [11]. So far, mutations in five ion channel genes (KCNQ1, HERG, SCN5A, KCNE1 and KCNE2) have been identified in patients with long QT syndrome. The mechanism of how these mutations alter electrophysiological stability is variable. Mutations may lead to a loss of channel function, alter protein trafficking, or result in dominant-negative properties [19,22]. Our present results demonstrate that the Andersen mutations rendered the KCNJ2 channel completely non-functional without affecting protein distribution in the cell membrane. Consistent with very recent observations in oocytes by others [5,11], the Andersen mutants suppressed wild-type KCNJ2 in a dominant-negative fashion when heterologously coexpressed in CHO cells. We observed a variable though overlapping degree of dominant-negative effect on the wild-type channel by the different mutants. This might at least in part explain the inconsistent and variable penetrance of disease mutations in afflicted individuals in vivo. However, similar to the other long QT channelopathies which exhibit only a weak genotype–phenotype correlation, additional yet unknown factors are likely to be involved [23].

Properties of cloned channel subunits studied in heterologous expression systems cannot readily be generalized to native channels and native tissue because of missing accessory subunits, local interacting proteins, differential glycosylation, or other influences of the cellular milieu. Thus, to also evaluate the effect of Andersen mutations in the native cellular environment we overexpressed the disease mutant KCNJ2-S136F in neonate rat cardiomyocytes using adenoviral gene transfer. In ventriculocytes Kir2 subunits are believed to underlie the major part of the inward rectifier current I_K1 which plays an important role in maintaining the resting membrane potential and in terminal cardiac repolarization [20,24–26]. Coexpression experiments indicated that the different subunits of the Kir2 family can form heteromultimeric complexes [5]. Consistent with dominant-negative suppression not only of the homomeric subunit Kir2.1, but also of Kir2.2 and Kir2.3 by the Andersen mutant, we indeed could demonstrate almost complete reduction of native I_K1 in KCNJ2-S136F-infected cardiocytes. This demonstrates that Kir2.x subunits are the major determinants of cardiac I_K1 in neonate rat ventricular cells supporting more recent results in guinea pigs and mice [27,28]. In guinea-pig and mouse ventricle a comparable reduction of I_K1 resulted in a prolongation of the QT interval and/or in spontaneous ventricular foci. Therefore, our observations now extend evidence that Andersen mutations by suppressing native I_K1 may predispose to arrhythmias and may lead to the phenotype of the long QT syndrome.

Given the multimerization properties of Kir2 subunits, it seems plausible that mutations of Kir2.2 or Kir2.3 might also underlie an Andersen-like syndrome. Indeed, in approximately 30% of patients fulfilling the diagnostic criteria of Andersen's syndrome no mutations of the KCNJ2 gene could be demonstrated [11]. In any event, since surprisingly in Kir2.2^−/− knock-out mice only a 50% reduction of native I_K1 was observed [26], any further disease mutations should always also be evaluated in native tissue. Detailed molecular insight into the mechanisms of channelopathies might facilitate the rational design of gene therapy strategies to modify cardiac arrhythmogenesis in the future.

Acknowledgements

We thank Dr. Francisco Rivero for help with confocal laser scanning. We thank Nadine Henn for skillful technical assistance. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ho 2146/2-1), by Köln Fortune (107/2002), and by the Marga and Walter Boll-Stiftung.