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Cardiovascular Research 2004 63(3):520-527; doi:10.1016/j.cardiores.2004.02.015
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Copyright © 2004, European Society of Cardiology

Human cardiac inwardly rectifying current IKir2.2 is upregulated by activation of protein kinase A

Edgar Zitrona, Claudia Kieseckera, Sonja Lücka, Sven Kathöfera, Dierk Thomasa, Volker A.W Kreyeb, Johann Kiehna, Hugo A Katusa, Wolfgang Schoelsa and Christoph A Karle*,a

aDepartment of Cardiology, Medical University Hospital Heidelberg, Bergheimerstrasse 58, D-69115, Heidelberg, Germany
bDepartment of Physiology and Pathophysiology, Medical University Hospital Heidelberg, Germany

* Corresponding author. Tel.: +49-6221-5638630; fax: +49-6221-565515. Email address: christoph1_karle{at}med.uni-heidelberg.de

Received 10 November 2003; revised 25 January 2004; accepted 18 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: The cardiac inwardly rectifying potassium current IK1 and its molecular correlates Kir2.1 and Kir2.2 play an important role in cardiac repolarisation and in the pathogenesis of hereditary long-QT syndrome (LQTS-7). Protein kinases A (PKA) and C (PKC) are key enzymes in adrenergic signal transduction, inducing arrhythmias in heart disease. This study investigated the regulation of Kir2.2 (KCNJ12) by PKA. Methods: Cloned Kir2.2 channels were expressed heterologously in Xenopus oocytes and currents were measured with the double-electrode voltage–clamp technique. Results: After activation of PKA by forskolin (100 µmol/l) or Ro-20-1724 (100 µmol/l), wild type currents at –120 mV were increased by 93.7% and 79.0%, respectively. Coapplication of the PKA inhibitor KT-5720 (2.5 µmol/l) attenuated this effect. No significant changes were apparent after mutation of the single PKA consensus site S430. In addition, removal of all four PKC consensus sites in Kir2.2 induced a phorbolester-mediated current increase which could be suppressed by PKA inhibitors H-89 (50 µmol/l) and KT-5720 (2.5 µmol/l). Conclusions: This study demonstrates antagonistic effects of PKA and PKC in the regulation of Kir2.2. Phosphorylation by PKC has been shown to cause an inhibition of Kir2.2 currents, whereas activation of PKA leads to current upregulation.

KEYWORDS Arrhythmia (mechanisms); K-channel; Protein kinase A; Protein kinase C; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
The inwardly rectifying potassium current IK1 plays a significant role in cardiac electrophysiology. IK1 is of major importance in the terminal phase of repolarisation and it is essential to maintain the resting membrane potential (RMP) of cardiomyocytes [1,2]. Near RMP, the ventricular IK1 conductance is much larger than that of any other current except for IK(ATP) which has a low activity under normoxic conditions [1,2]. Therefore, it is likely that physiological modulation of IK1 will have significant effects on excitability [1,2].

Clinically, IK1 has been found to be downregulated in electrophysiological remodeling due to heart failure [3]. In Andersen's syndrome, a recently described channelopathy based on mutations in Kir2.1 potassium channels, which contribute to IK1, QT prolongation and ventricular arrhythmias, have been observed [4]. Complex ventricular ectopy and polymorphic ventricular tachycardia were often noticed in patients with Andersen's syndrome whereas torsades de pointes and ventricular fibrillation were rare [5]. These cardiac manifestations are considered to be a consequence of reduced IK1 current density and have been classified as a new type of hereditary long QT syndrome (LQT-7) [5].

To date, the exact molecular composition of IK1 is still unclear. There is growing evidence that heteromeric assembly of Kir2.1 (KCNJ2) and Kir2.2/Kir2.1b (KCNJ12) potassium channels underlies a main part of the current [6–10]. Knockout mice lacking the Kir2.1 gene exhibited no measurable IK1 current whereas IK1 was reduced by almost 50% after Kir2.2 knockout [6]. In guinea pig heart, Kir2.2 appears to be the main contributor to IK1 [7]. It has been demonstrated that Kir2.1 and Kir2.2 form heteromeric channels in vitro and in rabbit cardiomyocytes [8,9]. Based on comparative studies of barium blocking properties, it has recently been suggested that human IK1 mainly results from heteromultimer formation among Kir2.1 and Kir2.2 [10].

Protein kinases have been found in almost all cell membranes and it has been demonstrated that they regulate ion channels both in native tissue and in expression systems [11–15]. Protein kinase A (PKA) and protein kinase C (PKC) are serine/threonine kinases which are involved in important pathways such as adrenergic signal transduction and the development of cardiac hypertrophy [16,17]. Modulation of IK1 by adrenergic receptors with consequent activation of PKA and PKC has repeatedly been demonstrated in human cardiomyocytes [18,19]. However, the molecular mechanisms underlying these regulatory effects have not been elucidated yet.

As Kir2.1 and Kir2.2 are considered to be the most relevant channels contributing to native IK1, studies focussing on interactions of PKA and PKC with cloned channels are most promising to provide further insights. Several studies have investigated regulatory effects of protein kinases on Kir2.1 channels with contradictory results [14,15,20–22]. We have demonstrated that protein kinase C has inhibitory effects on Kir2.2 currents via a direct phosphorylation of the channel protein likely to be relevant in vivo [11]. In this previous report, we suggested that Kir2.2 may be called Kir2.1b as it is closely related to Kir2.1 but displays different reactions to PKC activation [11]. However, we have decided to return to using the term Kir2.2 in line with standard nomenclature in order to avoid misunderstanding.

To date, interactions of protein kinase A with Kir2.2 channels have not been investigated. As endogenous protein kinases are present in Xenopus oocytes, they are a suitable expression system for research on the regulation of cloned channels [11,12,14,21,22]. Therefore, in this study, we investigated regulatory effects of protein kinase A on cloned Kir2.2 channels expressed in Xenopus oocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Solutions and drug administration
Two-microelectrode voltage clamp measurements of Xenopus oocytes were performed in a low K+ solution containing (in mM) 5 KCl, 100 NaCl, 1.5 CaCl2, 2 MgCl2 and 10 HEPES (pH 7.4). Current and voltage electrodes were filled with 3 M KCl solution. All measurements were carried out at room temperature (20 °C) as described previously [11]. Phorbol-12-myristate-13-actetate (PMA; Calbiochem, Germany), forskolin (Calbiochem) and Ro-20-1724 (Calbiochem) were dissolved in DMSO to a stock solution of 10 mmol/l and stored at –20 °C. On the day of experiments, aliquots of the stock solution were diluted to the desired concentrations with the bath solution. The maximum concentration of DMSO [0.1% (v/v)] in the bath had no effect on the measured currents.

2.2. Electrophysiology and data analysis
The two-microelectrode voltage clamp configuration was used to record currents from Xenopus laevis oocytes as described previously [11]. No leak subtraction was performed during the experiments. Only recordings with <10% leak current were considered for data analysis. Statistical data are presented as mean±standard error (S.E.M.). Statistical significance was evaluated using the paired and unpaired Student's t-test. Differences were considered to be significant if P<0.05 and highly significant if P<0.01.

2.3 Mutagenesis and expression of Kir2.2 channels in Xenopus oocytes
The Kir2.2 clone (GenBank accession no. L36069 [GenBank] ) was a kind gift from B.A. Wible (Cleveland, OH). Complementary RNA was prepared as described in Ref. [11]. Amino acid mutations were generated by polymerase chain reaction (PCR) with synthetic mutant oligonucleotide primers using the QuikChange site-directed mutagenesis kit (Stratagene, Germany) as described previously [11]. All mutations were verified by sequencing (SeqLab Goettingen, Germany). Injection of RNA (50 to 500 ng/µl) into stage V and VI defolliculated oocytes was performed using a Nanoject automatic injector (Drummond, Broomall, USA). The volume of injected cRNA solution was 50 nl per oocyte, and measurements were made 2 to 7 days after injection. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Activation of protein kinase A leads to an increase of Kir2.2 currents
A standardised voltage protocol was used in all measurements of this study to elicit characteristic inwardly rectifying Kir2.2 currents: test pulses to potentials ranging from –120 to +40 mV were applied in 10 mV increments (400 ms). The holding potential was –80 mV. Current traces from a representative experiment under control conditions are displayed in Fig. 1A. At potentials below the potassium reversal potential of approximately –80 mV under the given experimental conditions, Kir2.2 channels generated large inward currents. Voltage steps to more positive potentials did not elicit significant outward currents due to strong inward rectification typical for Kir2.2. In order to quantify increases and decreases of Kir2.2 currents, inward current amplitudes at –120 mV were measured and compared. Under control conditions, i.e., during perfusion with the bath solution, a run-up of Kir2.2 currents to 114.8±2.9% of the initial values was observed in 40 min (n=12).


Figure 1
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  		Fig. 1 Human Kir2.2 currents are activated by protein kinase A. Original Kir2.2 current traces from representative experiments are displayed in panels (A), (B), (D), (E). The first measurement was obtained under control conditions (A, D) and the second after 40 min of superfusion with protein kinase A activators forskolin (B) and Ro-20-1724 (D). Both substances induced a marked increase of Kir2.2 currents. Corresponding normalised current–voltage curves from all measurements are plotted in panels (C) and (F). Protocol: holding potential –80 mV; test pulses from –120 to +40 mV in 10 mV increments (400 ms).

 
In order to exclude a contribution of endogenous Xenopus oocyte channels to the measured currents, we also tested the effects of the voltage protocol described above on uninjected oocytes. In these native oocytes, no significant inward currents could be observed (n=6).

We chose a pharmacological approach to investigate the effects of protein kinase A (PKA) on Kir2.2 channels. As PKA activity depends on cyclic AMP (cAMP), stimulation of PKA can be induced indirectly by substances which increase the intracellular cAMP concentration. We used forskolin, an activator of adenylate cyclase [12,23], and Ro-20-1724, a specific inhibitor of phosphodiesterase IV [12,24].

First we investigated the effects of forskolin on Kir2.2 currents. After having obtained a control measurement (Fig. 1A), the cell was superfused with forskolin (100 µmol/l) continuously. After a wash-in period of 40 min, a final measurement was recorded (Fig. 1B). Forskolin induced a marked increase of Kir2.2 inward currents to 193.7±19.7% (n=6). Biophysical current properties were not affected (Fig. 1C).

In another set of experiments, the phosphodiesterase IV inhibitor Ro-20-1724 was applied under identical conditions. Typical recordings under control conditions and after exposure to Ro-20-1724 are displayed in Fig. 1D and E. A marked activation of Kir2.2 currents was observed resulting in an increase of inward currents to 179.0±16.3% (n=6). Like forskolin, Ro-20-1724 did not induce changes of biophysical current properties (Fig. 1F).

After exposure of the cells to forskolin or Ro-20-1724, the cells were superfused with the bath solution for additional 20 min. Within this washout period, currents increased by 21.0±6.6% in the forskolin group and by 23.8±5.1% in the Ro-20-1724 group, respectively. Thus, the effects of forskolin and Ro-20-1724 were not reversible upon washout.

In order to monitor the time course of effect, measurements were repeated at intervals of 5 min during the observation period of 40 min. In Fig. 2A, relative current amplitudes of representative experiments under control conditions and during exposure to forskolin (100 µmol/l) are plotted as function of time. The increase of currents induced by forskolin was markedly larger than the run-up observed during perfusion with the bath solution. Although even after 40 min, a steady state was not reached, a marked effect could already be observed. As it is technically difficult to perform electrophysiological experiments with Xenopus oocytes with longer wash-in periods, 40 min were chosen as common observation period of the experiments. Statistical analysis of the results is shown in Fig. 2B. Both the effects of forskolin (p=4.04 x 10–5) and of Ro-20-1724 (p=6.21 x 10–5) were significantly different from the current run-up under control conditions.


Figure 2
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  		Fig. 2 Time course and statistical analysis of the effects of forskolin and Ro-20-1724 on Kir2.2 currents. In panel (A), time course of the effect of forskolin on Kir2.2 currents is plotted in comparison to the run-up observed under control conditions. Data from typical experiments are shown. Statistical analysis is summarised in panel (B). Both Ro-20-1724 (100 µmol/l) and forskolin (100 µmol/l) induced a marked increase of Kir2.2 currents with a significant difference to the control experiments indicated by asterisks. Coapplication of KT-5720 attenuated the effects significantly (indicated by darker colour of columns).

 
The specific PKA-inhibitor KT-5720 [24] was used to gain additional evidence for the role of protein kinase A in mediating the current increase. KT-5720 at a concentration of 2.5 µmol/l was coapplied with forskolin (100 µmol/l) and Ro-20-1724 (100 µmol/l), respectively. Experiments were performed under conditions identical to those described above. KT-5720 attenuated the effects of both forskolin and Ro-20-1724, resulting in an increase of currents to 138.7±11.7% (n=5) and 135.4±5.3% (n=5), respectively (Fig. 2B). These results were significantly different from those observed after application of forskolin and Ro-20-1724 without KT-5720 (p=0.048 and p=0.045, respectively).

3.2. Activation of Kir2.2 channels lacking PKC consensus sites by phorbol ester PMA is mediated by protein kinase A
In the amino acid sequence of Kir2.2 channels, four consensus sites for protein kinase C-dependent phosphorylation (thr-38, ser-64, thr-353, ser-357) and one site for protein kinase A (ser-430) can be identified [25]. Their localisation within the channel subunit is schematically visualised in Fig. 3.


Figure 3
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  		Fig. 3 Protein kinases A and C consensus sites in Kir2.2 channels. Sequence analysis of Kir2.2 revealed multiple-consensus sites for protein kinase phosphorylation [11,25]: four sites for PKC phosphorylation (T38, S64, T353, S357) and one site for PKA phosphorylation (S430).

 
We reported previously that protein kinase C has inhibitory effects on Kir2.2 channels by direct phosphorylation of the channel protein at the PKC consensus sites [11]. In Kir2.2 wild type channels, the phorbol ester PMA, an unspecific activator of protein kinases, at a concentration of 100 nmol/l, leads to a reduction of relative currents to 28.8±8.9% of the initial values [11]. Surprisingly, in mutant Kir2.2-4M channels lacking all four PKC consensus sites, PMA induced an increase of currents to 171.8±15.1% during an observation period of 30 min (n=6) [11]. Representative recordings from these experiments are displayed in Fig. 4. In our previous study, we did not investigate the basis of this effect.


Figure 4
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  		Fig. 4 Activation of Kir2.2-4M channels by PMA is mediated by protein kinase A. In mutant Kir2.2 channels lacking all four PKC consensus sites, unspecific protein kinase activator PMA induced an increase of currents. Measurements from representative experiments and normalised current–voltage curves from all measurements are displayed in panels (A), (B), (C). This effect could be suppressed by coapplication of protein kinase A inhibitors H-89 and KT-5720 which points to an involvement of PKA. Results are compared in panel (D). Protocol: holding potential –80 mV; test pulses from –120 to +40 mV in 10 mV increments (400 ms).

 
Interestingly, PMA is not only a potent activator of protein kinase C but also stimulates adenylate cyclase resulting in an increase of protein kinase A activity [12]. Given our findings indicating an activation of Kir2.2 channels by protein kinase A, we sought to test whether this regulatory pathway might also be involved in the antagonistic effects of PMA on wild type and mutant Kir2.2 channels. Two specific inhibitors of protein kinase A were chosen for further experiments: H-89 [23] and KT-5720 [24]. Both were coapplied with 100 nmol/l PMA: H-89 at a concentration of 50 µmol/l and KT-5720 at 2.5 µmol/l, respectively. Kir2.2 currents were recorded as described previously in this study. An observation period of 30 min was chosen to allow comparison with the results in Ref. [11].

Statistical analysis of the results is plotted in Fig. 4D. Both PKA inhibitors markedly attenuated the effect of PMA: relative current increased to 120.1±5.4% (n=5) after coapplication of H-89 and to 121.4±2.8% (n=6) after coapplication of KT-5720. These values were significantly different from the effect of PMA (p=0.02 and p=0.01, respectively). Thus, protein kinase A is likely to be involved in the activation of Kir2.2-4M channels by PMA.

3.3. Kir2.2 consensus site ser-430 is not essential for protein kinase A-mediated activation
In order to elucidate the molecular mechanism of the observed effect of protein kinase A on Kir2.2 channels, we studied the significance of the only consensus site for PKA phosphorylation in the Kir2.2 amino acid sequence, ser-430. We generated Kir2.2-S430A mutant channels by replacing serine with alanine, which cannot be phosphorylated, and therefore inactivates the consensus site. Under control conditions, Kir2.2-S430A displayed biophysical properties identical to wild type channels (Fig. 5).


Figure 5
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  		Fig. 5 PKA consensus site S430 is not essential for PKA-mediated activation. Original recordings from typical experiments and normalised current–voltage curves from all measurements are displayed in panels (A), (B), (C). Kir2.2-S430A mutant channels exhibited biophysical current properties indistinguishable from wild type channels (A, C). Forskolin induced an increase of currents comparable to the effect observed in wild type channels (B). Results of experiments with forskolin and Ro-20-1724 in S430A and wild type channels are compared in panel (D). No significant difference was observed. Protocol: holding potential –80 mV; test pulses from –120 to +40 mV in 10 mV increments (400 ms).

 
Experiments with Kir S430A were performed under conditions identical to the wild type as described above. Forskolin and Ro-20-1724 were applied at a concentration of 100 µmol/l for 40 min. Both substances lead to a marked increase of Kir2.2-S430A currents which was comparable to the effects on wild type channels. Ro-20-1724 induced an increase to 152.6±7.1% (n=5) and forskolin to 172.8±12.8% (n=7). As in the experiments with Kir2.2 wild type channels, reversibility of the effect upon washout was tested by superfusion of the cells with the bath solution for 20 min after exposure to forskolin. During the washout period, inward currents increased by 12.7±3.4% (n=7). Thus, the effect of forskolin on Kir2.2-S430A currents was not reversible upon washout.

Results are summarised and compared statistically in Fig. 5D. The effects of Ro-20-1724 and forskolin on Kir2.2-S430A were not significantly different from those on Kir2.2 wild type (p=0.11 and p=0.38, respectively). Therefore, PKA consensus site ser-430 is probably not essential for the upregulation of Kir2.2 currents by protein kinase A.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
4.1. Antagonistic regulation of human Kir2.2 channels by protein kinases A and C
In this study, we have demonstrated that activation of protein kinase A induces an increase of human cardiac inwardly rectifying Kir2.2 currents in the Xenopus oocyte expression system. Indirect PKA activation by forskolin and by Ro-20-1724 lead to a marked activation of Kir2.2 channels without significant changes in biophysical current properties. These effects could be attenuated by coapplication of the specific PKA inhibitor KT-5720.

Under control conditions, a mild run-up of Kir2.2 currents was observed which has already been described previously [11]. The cause of this run-up is unclear [11]. However, a similar phenomenon has been described for other channels expressed in Xenopus oocytes such as HERG channels [26]. Therefore, the run-up may be due to specific properties of the Xenopus oocyte expression system [26].

Current increase induced by PKA activation did not reach a plateau within an observation time of 40 min. During a following washout period of 20 min, the effect was not reversible. In contrast, increase of currents continued to a reduced extent. These results would fit in with a slow kinetic of the intracellular signal transduction pathways involved in mediating the effect. However, there also are other possible explanations. It may be that the effects do not exhibit a pharmacological saturation and do not reach a plateau as a consequence. Furthermore, the kinetic we observed may be due to unspecific effects related to the expression system.

Kir2.2 channels exhibit only one typical PKA consensus site at ser-430. Inactivation of this consensus site in Kir2.2-S430A mutant channels did not affect current activation by protein kinase A. Therefore, ser-430 probably does not play a role in mediating the effect. A likely explanation for this finding may be a phosphorylation of the channel protein at an atypical phosphorylation site which does not fit into the classical consensus pattern and cannot be detected by sequence analysis. This has been reported to be the case in the inhibitory regulation of Kir2.3 channels by protein kinase C via a phosphorylation at residue thr-53 which is not part of a consensus site [22]. Another possibility is the regulation of the channel independent of phosphorylation: for example, the effects of PKC on HERG channels have been demonstrated to be independent of phosphorylation and have therefore been attributed to the activation of intermediate factors of signal transduction [27]. However, the molecular mechanism of the observed effect remains unclear and has to be elucidated by further studies.

In a previous study, we found that protein kinase C has inhibitory effects on Kir2.2 channels (named Kir2.1b in our report) via a direct phosphorylation of the channel protein [11]. This regulation was observed in human cardiomyocytes and investigated in detail in the Xenopus oocyte expression system using mutated Kir2.2 channels [11]. However, at least 12 different PKC isoforms have been described of which 5–7 are expressed in human myocardium [17]. Data on PKC isoform expression in Xenopus oocytes is not available to date. Thus, PKC isoforms active in Xenopus oocytes and in cardiomyocytes and the resulting phosphorylation patterns may be different. As a consequence, it may be difficult to transfer the results concerning PKC-dependent signal transduction obtained in Xenopus oocytes to human cells. Further studies are needed to clarify which PKC isoforms are involved in this regulation and what their role in human myocardium may be.

Thus, Kir2.2 channels are antagonistically regulated by protein kinases A and C according to results obtained in Xenopus oocytes. This is illustrated in Fig. 6: under control conditions, a run-up of Kir2.2 currents to 114.8±2.9% was observed (n=12). Activation of PKC by thymeleatoxin (100 nmol/l) induced a reduction of currents to 18.6±0.4% (n=5) [11] whereas activation of PKA by forskolin (100 µmol/l) led to an increase of currents to 193.7±19.7% (n=7). If this antagonistic regulation is also active in human myocardium, it may represent an interesting physiological mechanism modulating repolarisation and excitability of cardiomyocytes.


Figure 6
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  		Fig. 6 Antagonistic regulation of human Kir2.2 channels by protein kinases A and C. Results from experiments with activators of protein kinases A and C are shown to illustrate antagonistic regulation. PKC activator thymeleatoxin (100 nmol/l) leads to a reduction of Kir2.2 currents to 18.6±0.4% of the initial values (middle column labelled "PKC") [11]. In contrast, forskolin (100 µmol/l) induced an increase of Kir2.2 currents to 193.7±19.7% (right column labelled "PKA").

 
4.2. Differential effects of protein kinase A on Kir2.1 and Kir2.2 channels
Effects of protein kinase A on cloned Kir2.1 channels have been investigated in several studies with contradictory results. Fakler et al. [14] observed an activation of Kir2.1 in inside-out-patches from Xenopus oocytes. Wischmeyer and Karschin [15] found a marked PKA-dependent inhibition of Kir2.1 channels expressed in COS cells mediated by a phosphorylation at ser-425. In contrast, Dart and Leyland [20] studied Kir2.1 channels expressed in CHO and HEK cells and found a mild activation of the currents by PKA which was enhanced after coexpression of AKAP 79. To date, there is no sufficient explanation for these contradictory observations [1].

Human Kir2.1 and Kir2.2 channels exhibit a sequence homology of almost 70% [25]. They also share a homologous PKA consensus site located at ser-425 (Kir2.1) and ser-430 (Kir2.2), respectively. Interestingly, protein kinase A may have differential effects on these two closely related channels and the PKA consensus site may be physiologically active in Kir2.1, whereas it is not relevant in Kir2.2 according to our findings.

4.3. Potential significance in vivo
From a theoretical point of view, an activation of Kir2.2 channels and a consequent increase of IK1 current in vivo would be expected to lead to a shortening of the myocardial action potential and a stabilisation of the resting membrane potential [1]. This has already been demonstrated in animal models and in computer simulations showing a reduction of the risk of ventricular arrhythmias as a consequence [2,28].

Regulation of native IK1 by protein kinase A has not been studied extensively. Koumi et al. [18] demonstrated an inhibition of IK1 by PKA-dependent pathways in human cardiomyocytes. The divergence of this finding and our results may be due to several factors illustrating potential limitations of our study. As an inhibition of Kir2.1 channels by PKA has been described [15], this effect might be predominant in vivo and outweigh the activation of Kir2.2 channels. Furthermore, heteromeric assembly of Kir2 channels is not completely understood yet and may also have consequences on the sensitivity of the channels towards protein kinase A.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
This is the first study demonstrating an activation of human cardiac Kir2.2 channels by protein kinase A. These results point to an antagonistic regulation of Kir2.2 channels by protein kinases A and C which may contribute to the modulation of cardiac repolarisation and excitability. Our findings provide further insights into the molecular basis of the adrenergic regulation of IK1 in the heart.


   Acknowledgements
 
The excellent technical assistance of Ramona Bloehs and Klara Gueth is gratefully acknowledged. This work was supported by grants of the Deutsche Forschungsgemeinschaft Ka 1714/1-1 to Dr. Karle and Ki 6631/1 to Dr. Kiehn. E. Zitron was supported by the German National Scholarship Foundation. Dr. Thomas was supported by grants from the University of Heidelberg (AiP+F), the Novartis Foundation and the Foundation Cardiology 2000 (Forssmann-Scholarship).


   Notes
 
Time for primary review 24 days


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

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  5. Tristani Firouzi M, Jensen J.L, Donaldson M.R, et al. Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome). J. Clin. Invest. (2002) 110:381–388.[CrossRef][ISI][Medline]
  6. Zaritsky J.J, Redell J.B, Tempel B.L, Schwarz T.L. The consequences of disrupting cardiac inwardly rectifying K(+) current (I(K1)) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes. J. Physiol. (2001) 533:697–710.[Abstract/Free Full Text]
  7. Liu G.X, Derst C, Schlichthörl G, et al. Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea pig cardiomyocytes. J. Physiol. (2001) 532:115–126.[Abstract/Free Full Text]
  8. Preisig-Müller R, Schlichthörl G, Goerge T, et al. Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome. Proc. Natl. Acad. Sci. U. S. A. (2002) 99:7774–7779.[Abstract/Free Full Text]
  9. Zobel C, Cho H.C, Nguyen T.T, et al. Molecular dissection of the inward rectifier potassium current (IK1) in rabbit cardiomyocytes: evidence for heteromeric coassembly of Kir2.1 and Kir2.2. J. Physiol. (2003) 550:365–372.[Abstract/Free Full Text]
  10. Schram G, Pourrier M, Wang Z, White M, Nattel S. Barium block of Kir2 and human cardiac inward rectifier currents: evidence for subunit-heteromeric contribution to native currents. Cardiovasc. Res. (2003) 59:328–338.[Abstract/Free Full Text]
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