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Cardiovascular Research 2007 74(3):426-437; doi:10.1016/j.cardiores.2007.02.009
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Copyright © 2007, European Society of Cardiology

Differential phosphorylation-dependent regulation of constitutively active and muscarinic receptor-activated IK,ACh channels in patients with chronic atrial fibrillation

Niels Voigta, Adina Friedricha, Manja Bockb, Erich Wettwera, Torsten Christa, Michael Knautc, Ruth H. Strasserb, Ursula Ravensa and Dobromir Dobreva,*

aDepartment of Pharmacology and Toxicology, Dresden University of Technology, Dresden, Germany
bDepartment of Cardiology, Dresden University of Technology, Dresden, Germany
cDepartment of Cardiosurgery, Dresden University of Technology, Dresden, Germany

* Corresponding author. Fetscherstr. 74, 01307 Dresden, Germany. Tel.: +49 351 4586279; fax: +49 351 4586315. Email address: dobrev{at}rcs.urz.tu-dresden.de

Received 21 September 2006; revised 17 January 2007; accepted 5 February 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Objective: In chronic atrial fibrillation (cAF) the potassium current IK,ACh develops agonist-independent constitutive activity. We hypothesized that abnormal phosphorylation-dependent regulation underlies the constitutive IK,ACh activity.

Methods: We used voltage-clamp technique and biochemical assays to study IK,ACh regulation in atrial appendages from 61 sinus rhythm (SR), 11 paroxysmal AF (pAF), and 33 cAF patients.

Results: Compared to SR basal current was higher in cAF only, whereas the muscarinic receptor (2 µmol/L carbachol)-activated IK,ACh was smaller in pAF and cAF. In pAF the selective IK,ACh blocker tertiapin abolished the muscarinic receptor-activated IK,ACh but excluded agonist-independent constitutive IK,ACh activity. Blockade of type-2A phosphatase and the subsequent shift to increased muscarinic receptor phosphorylation (and inactivation) reduced muscarinic receptor-activated IK,ACh in SR but not in cAF, pointing to an impaired function of G-protein-coupled receptor kinase. Using subtype-selective kinase inhibitors we found that in SR the muscarinic receptor-activated IK,ACh requires phosphorylation by protein kinase G (PKG), protein kinase C (PKC), and calmodulin-dependent protein kinase II (CaMKII), but not by protein kinase A (PKA). In cAF, constitutive IK,ACh activity results from abnormal channel phosphorylation by PKC but not by PKG or CaMKII, whereas the additional muscarinic receptor-mediated IK,ACh activation occurs apparently without involvement of these kinases. In cAF, the higher protein level of PKC{varepsilon} but not PKC{alpha}, PKCβ1 or PKC{delta} is likely to contribute to the constitutive IK,ACh activity.

Conclusions: The occurrence of constitutive IK,ACh activity in cAF results from abnormal PKC function, whereas the muscarinic receptor-mediated IK,ACh activation does not require the contribution of PKG, PKC or CaMKII. Selective drug targeting of constitutively active IK,ACh channels may be suitable to reduce the ability of AF to become sustained.

KEYWORDS Fibrillation; Atrium; Potassium channels; Kinases; Phosphatases


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Atrial fibrillation (AF) is the most frequent arrhythmia in the clinical setting. It is accepted that electrical and structural remodeling including abbreviated action potentials and refractoriness, hypertrophy and fibrosis promote the existence of reentry circuits and the progression of AF [1]. Studies in animal models and in patients with chronic AF (cAF) suggest that the G-protein-gated potassium current IK,ACh plays a crucial role in atrial arrhythmogenesis [2–6].

We have shown that the background inward rectifier potassium current IK1 is higher in cAF and was associated with smaller muscarinic receptor-mediated IK,ACh activation [3–5]. Interestingly cAF patients exhibit agonist-independent constitutive IK,ACh activity that contributes to the enhanced basal inward rectifier current [5]. In dogs a corresponding constitutively active IK,ACh-like current exists which is up-regulated in response to atrial tachypacing [6,7]. Blockade of this current by the selective IK,ACh- blocker tertiapin resulted in action potential prolongation and suppression of inducible AF episodes [6]. Thus, identifying the culprit abnormalities in IK,ACh regulation may lead to novel therapeutic targets for AF prevention and therapy.

The molecular basis of constitutively active IK,ACh in cAF is unknown. At the single channel level, agonist-independent IK,ACh activity in cAF results from higher channel open probability without changes in amplitudes and open times. Earlier studies suggest that agonist-independent activation of IK,ACh requires ATP [8–10]. Thus modified phosphorylation of IK,ACh and regulatory proteins may contribute to constitutively active IK,ACh in cAF. The IK,ACh channel forms a macromolecular complex, allowing for local IK,ACh regulation. The GIRK1 channel complex may comprise the catalytic subunits of protein kinases A (PKA) and C (PKC), the calmodulin-regulated protein kinase II (CaMKII), and the type-1 and type-2A protein phosphatases PP1 and PP2A [11]. The kinase/phosphatase signaling in the cellular microdomains of cAF patients is inhomogeneous resulting in increased, decreased or normal phosphorylation levels of proteins despite enhanced total PP1 and PP2A activity [12–14]. Thus, the quantitative and qualitative composition of the macromolecular channel complex may change during cAF resulting in abnormal phosphorylation-dependent IK,ACh regulation.

The present study tested the hypothesis that alterations in phosphorylation-dependent channel regulation may contribute to constitutive IK,ACh activity in cAF.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
2.1 Human samples
The study was approved by the local ethics committee of the university (No: EK790799) and each patient gave written informed consent. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–4).

Right atrial appendages were obtained from 61 patients with SR, 11 patients with paroxysmal AF (pAF) and 33 patients with cAF (cAF>6 months, Table 1 and supplementary data).


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  		Table 1 Characteristics of patients used for the electrophysiological experiments

 
2.2 Electrophysiological recordings
Atrial myocytes were isolated using our previous protocol [15] and were suspended in storage solution (in mmol/L: KCl 20, KH2PO4 10, glucose 10, K-glutamate 70, β-hydroxybutyrate 10, taurine 10, EGTA 10, albumin 1, pH=7.4). Membrane currents were measured with voltage-clamp technique. ISO-2 software (MFK) was used for data acquisition and analysis.

Borosilicate glass microelectrodes had tip resistances of 2–5 M{Omega} when filled with pipette solution (in mmol/L: K-aspartate 80, NaCl 8, KCl 40, Mg-ATP 5, EGTA 2, GTP-Tris 0.1, HEPES 10, pH=7.4). Myocytes were superfused with a solution containing (in mmol/L): NaCl 120, KCl 20, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, pH=7.4 at 22–24 °C. Seal resistances were 4–8 G{Omega}. Series resistance and cell capacitance were compensated. Agonist-independent basal current was measured by applying a ramp pulse from –100 to +40 mV (Fig. 1A). Agonist-inducible IK,ACh was stimulated with carbachol (CCh, 2 µmol/L) in the absence and presence of selective inhibitors of protein kinase G (PKG; KT5823, 1 µmol/L), PKC (bisindolylmaleimide-I [BIM-I] and its inactive form bisindolylmaleimide-V [BIM-V], 0.1 µmol/L each), CaMKII (KN-93 and its inactive form KN-92, 0.2 µmol/L each), protein kinase A (PKA; KT5720, 1 µmol/L), PP1/PP2A (okadaic acid, 1 µmol/L) and type-2B protein phosphatase calcineurin (PP2B; cyclosporin A, 10 µmol/L). All drugs were from Calbiochem.


Figure 1
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  		Fig. 1 Inward rectifier currents in atrial myocytes from SR and cAF. A, Original recording of basal current and response to 2 µmol/L carbachol (CCh;IK,ACh defined as CCh-sensitive current). B, Time course of IK,ACh. During activation the initial increase ("Peak") faded ("rapid desensitisation") to a quasi steady-state level ("QSS"). Arrows: time point of measurements for D. C, Basal current and CCh-activated IK,ACh at "Peak" and "QSS" in SR, pAF and cAF. D, Basal resting membrane potential (RMP) and CCh-induced hyperpolarisation. Numbers indicate myocytes/patients. *P<0.05 vs. corresponding values in SR and pAF; #P<0.05 vs. CCh in SR.

 
Ba2+ (1 mmol/L) was applied in each myocyte and the currents were analyzed after subtraction of the resulting leak current. The myocytes were superfused with Tyrode's solution, and the drugs were applied via an additional rapid solution exchange system (ALA Scientific Instruments, Long Island, NY, USA). To control for myocyte-size variability, the currents are expressed as densities (pA/pF).

2.3 Western blot analysis
Protein levels of calsequestrin (1:2500; Dianova), PKC{alpha} (1:1000), PKCβ1 (1:250), PKC{delta} (1:250), and PKC{varepsilon} (1:500; all Santa Cruz) were quantified in atrial homogenates by Western blotting as described [16]. Immunological signals were visualized with anti-IgG-horseradish-peroxidase and enhanced chemifluorescence (Amersham) and quantified using Quantity One software (BioRad).

2.4 Molecular analysis of the Gβ3 gene
Direct activation of IK,ACh is mediated by G-protein β{gamma}-subunits. The G-protein β3 subunit gene (GNB3) contains a C825T-polymorphism, whereby homozygous 825T-allele carriers exhibit larger basal inward rectifier K+ current possibly due to enhanced signal transduction. To exclude confounding by homozygous 825T-allele carriers, all patients were genotyped [15]. The patient sample includes homozygous and heterozygous C825-allele carriers only.

2.5 Statistical analysis
One-way ANOVAs were applied to determine the sources of K+-current and protein variation (SPSS version 12.0). Independent variables were rhythm status, selected clinical variables and medication (Table 1 and supplementary data). To test for interactions between rhythm status and clinical variables or medication, interaction terms were included in separate two-way ANOVA. Differences between group means for continuous data were compared by unpaired Student's t-test or by one-way ANOVA and Bonferroni multiple comparisons procedure. Frequency data were analyzed with {chi}2 statistics. Data are means±SEM. P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 
Significant differences between the groups were found for gender, age, coronary artery disease, valvular heart disease, hyperlipidemia and left atrial diameter. cAF patients more often received digitalis but less frequently lipid-lowering drugs. Diuretics were more often prescribed in pAF and cAF than SR (Table 1). With one-way ANOVAs, AF was the only predictor of current densities and protein levels (not shown).

3.1 Basal current and CCh-activated IK,ACh
Cell capacitances averaged 99.6±3.1 pF (n=108/48; myocytes/patients) for SR, 80.9±6.5 pF (n=23/11) for pAF and 109.3±5.3 pF (n=48/20) for cAF myocytes (P=N.S.). Typical current traces and current amplitudes at –100 mV in the time course of an experiment are shown in Fig. 1A,B. Basal currents in SR and pAF were similar (–12.8±0.7 pA/pF, n=108/48 vs. –14.1±1.5 pA/pF, n=23/11), but smaller than in cAF (–20.7±1.2 pA/pF, n=48/20; P<0.05; Fig. 1C) confirming previous results [3–5,17,18]. The smaller basal currents in SR and pAF are in accordance with the resting membrane potential (RMP) being less negative in SR and pAF than in cAF (–18.8±0.9 mV, n=86/48 and –15.6±1.6 mV, n=12/4 vs. –23.0±1.4 mV, n=48/20; P<0.05; Fig. 1D) [3,4,17].

Application of CCh resulted in rapid initial increase ("Peak") of IK,ACh followed by a decrease to a quasi steady-state level ("QSS") despite continuous presence of CCh ("desensitisation"; Fig. 1B) [5]. There was no difference in IK,ACh desensitisation between the groups (QSS/Peak ratio: SR=0.58±0.01 [n=108/48], pAF=0.59±0.05 [n=23/11], and cAF=0.56±0.09 [n=48/20]; P=N.S.). However, the CCh-activated Peak- and QSS-IK,ACh were smaller in pAF and cAF than in SR (Peak-IK,ACh: –5.1±0.9 pA/pF and –6.3±1.0 pA/pF vs. –11.0±1.0 pA/pF; QSS-IK,ACh: –2.5±0.4 pA/pF and –3.8±0.6 pA/pF vs. –6.4±0.6 pA/pF, P<0.05; Fig. 1C). The smaller CCh-activated IK,ACh resulted in less CCh-induced hyperpolarisation of RMP in cAF (–7.3±1.1 mV, n=48/20) and pAF (–7.7±1.9 mV, n=12/4) than in SR (–12.6±0.9 mV, n=86/48; Fig. 1D). With two-way ANOVA, test of interaction effects between coronary artery disease, valvular heart disease, hyperlipidemia, digitalis, diuretics, lipid-lowering drugs, gender, age or left atrial diameter and IK,ACh showed no significant interaction (not shown).

3.2 Effects of tertiapin on basal current and CCh-activated IK,ACh in pAF patients
In experiments with selective inhibitors, IK,ACh was stimulated twice (S1, S2) with 4 min CCh-free period in between. S1 served as internal control, S2 was measured in the presence of inhibitors [5]. Under control conditions IK,ACh was smaller during S2 than S1 suggesting incomplete recovery from desensitisation, but the degrees of IK,ACh desensitisation were similar between the groups (Fig. 2A).


Figure 2
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  		Fig. 2 Repeated activation of IK,ACh by carbachol. A, Activation of IK,ACh during 2 successive carbachol (CCh, 2 µM) applications (S1, S2, 4 min apart) in SR, pAF and cAF (left). Mean values of Peak- and QSS-IK,ACh and the corresponding ratio in SR, pAF and cAF (right). B, Concentration-dependent effects of tertiapin on the S2/S1 ratio of Peak-IK,ACh in pAF (left). Concentration-dependent block of basal current with tertiapin in pAF (right). Numbers indicate myocytes/patients.

 
In pAF, the selective IK,ACh channel blocker tertiapin (0.1–100 nM) during S2 reduced the S2/S1 ratio in a concentration-dependent manner without an effect on basal current (Fig. 2B) excluding constitutive IK,ACh activity.

3.3 Regulation of basal current and CCh-activated IK,ACh by protein phosphatases
Regulation of IK,ACh through phosphorylation is a dynamic process that reflects the actual balance between kinase and phosphatase activities at the various sites of signal transduction (Fig. 3). Upon addition of CCh during S1, direct binding of liberated Gβ{gamma}-subunits to GIRK1/GIRK4 causes rapid activation of IK,ACh (1) [10] followed by desensitisation, the fast phase of which (green) is associated with channel dephosphorylation (2) [19,20], whereas the intermediate phase (blue) involves progressive receptor phosphorylation by a G-protein-coupled receptor kinase (GRK) which uncouples the receptor from the G-protein (3) [21]. During the CCh-free period (4) channel phosphorylation and receptor dephosphorylation recover only incompletely leading to a smaller CCh-activated IK,ACh during S2 [5]. Thus, phosphorylation of muscarinic receptors or IK,ACh channels modulates IK,ACh in a reciprocal manner, with receptor phosphorylation decreasing and channel phosphorylation increasing current amplitude.


Figure 3
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  		Fig. 3 Working model of phosphorylation-dependent IK,ACh regulation in the atrium. Time course of a typical experiment and putative changes in the phosphorylation state of muscarinic receptors and IK,ACh channels. The balance between phosphorylated muscarinic receptor and IK,ACh channel is controlled by various kinases and phosphatases (bottom panel). Whereas channel phosphorylation increases IK,ACh, concomitant phosphorylation of muscarinic receptors reduces IK,ACh (right panel, top). See text for further details.

 
Since total PP1 and PP2A activity is higher in cAF than in SR [13,14] we investigated the contribution of phosphatases to the regulation of IK1, constitutively active and CCh-activated IK,ACh channels. Selective phosphatase inhibitors were applied 3 min before and during S2 (Fig. 4A). In SR, basal current was affected neither by the PP1/PP2A-inhibitor okadaic acid (1 µmol/L) nor by the calcineurin inhibitor cyclosporin A (10 µmol/L; Fig. 4A,B) and there was no change in RMP with either inhibitor (Fig. 4C). In contrast, inhibition of PP1 and PP2A led to a small but significant reduction of basal current in cAF (Fig. 4A,B), which was associated with a significant change of RMP leading to less negative potentials (Fig. 4C).


Figure 4
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  		Fig. 4 Effects of phosphatase inhibitors on basal current and CCh-activated IK,ACh. A, Original time course of CCh-activated IK,ACh and effect of okadaic acid (OA, 1 µmol/L) in both groups. B and C, Mean changes of basal current and RMP by OA and cyclosporin A (CsA, 10 µmol/L) in SR and cAF. D and E, Effects of OA and CsA on the S2/S1 ratio of Peak- and QSS-IK,ACh and on CCh-induced changes in RMP in either group. Numbers indicate myocytes/patients. *P<0.05 vs. corresponding values in SR or cAF.

 
Discriminating the contribution of phosphatases to IK,ACh is impeded by concomitant modulation of receptor phosphorylation (Fig. 3). PP2A (but not PP1 and calcineurin) is a component of the M2-receptor complex [11] and PP2A inhibition is expected to shift the balance in favor of receptor phosphorylation by GRK resulting in reduced IK,ACh activation (Fig. 3) [21]. In the presence of okadaic acid, CCh-activated Peak- and QSS-IK,ACh were ~35% lower in SR, whereas cyclosporin A was without effect (Fig. 4D). In cAF, however, okadaic acid did not affect CCh-activated IK,ACh suggesting potentially impaired function of GRK (Fig. 4D). The CCh-induced changes in RMP exhibited large inter- and intra-patient variability (Fig. 1D). Therefore, in analogy to the S2/S1 analysis of CCh-activated IK,ACh, the corresponding effects of CCh on RMP were expressed as the ratio of the CCh-induced changes in RMP during S1 and S2, i.e. the CCh-sensitive change in RMP ({delta}RMP) during S2 is divided by the corresponding value during S1 ({delta}RMP(S2)/{delta}RMP(S1)). In SR, the okadaic acid-associated decreases of CCh-activated IK,ACh resulted in corresponding changes in RMP (Fig. 4E).

3.4 Regulation of basal current and CCh-activated IK,ACh by protein kinases
In SR, the PKG-inhibitor KT5823 (1 µmol/L) reduced basal current by 3.9±1.1 pA/pF (n=9/4), though without significant changes in RMP (Fig. 5A–C). Since basal current in SR consists of IK1 only [5], our results suggest that IK1 requires channel phosphorylation by PKG. The PKC-inhibitor BIM-I (0.1 µmol/L), the CaMKII-inhibitor KN-93 (0.2 µmol/L) and the PKA-inhibitor KT5720 (1 µmol/L) had no effect on basal current in SR (Fig. 5A,B). Accordingly, RMP remained unchanged (Fig. 5C).


Figure 5
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  		Fig. 5 Effects of kinase inhibitors on basal current and CCh-activated IK,ACh in SR. A, Effects of PKG, PKC, CaMKII and PKA inhibitors (1 µmol/L KT5823, 0.1 µmol/L BIM-I, 0.2 µmol/L KN-93 and 1 µmol/L KT5720, respectively) on basal current. BIM-V (0.1 µmol/L) and KN-92 (0.2 µmol/L) served as negative controls. B and C, Effects of kinase inhibitors on basal current and RMP, respectively. Numbers indicate myocytes/patients. *P<0.05 vs. control.

 
Basal current in cAF involves IK1 and constitutively active IK,ACh [5]. Thus any change in basal current could result from impaired regulation of either channel. KT5823 decreased basal current in cAF in the same order of magnitude as in SR confirming the positive regulation of IK1 by PKG (Fig. 6A,B). KN-93 and its inactive analog had no effect on basal current or RMP. BIM-I, but not its negative analog, reduced basal current by 3.4±0.8 pA/pF (n=7/3) and changed RMP to more positive potentials (Fig. 6A–C).


Figure 6
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  		Fig. 6 Effects of kinase inhibitors on basal and CCh-activated IK,ACh in cAF. Same layout as in Fig. 5.

 
Lack of contribution of CaMKII, PKC, PKG and PKA to muscarinic receptor phosphorylation allows to investigate the contribution of these kinases to IK,ACh regulation without confounding effects on muscarinic receptor function (Fig. 3) [11,22]. In SR, KT5823, BIM-I and KN-93 reduced Peak- but not QSS-IK,ACh. The inactive analogs were without effect (Fig. 7A). The kinase inhibitor-associated decreases of Peak-IK,ACh were paralleled by corresponding changes in RMP (Fig. 7B). Signal transduction via muscarinic receptors does not involve activation of PKA [23] and accordingly KT5720 did not affect CCh-activated IK,ACh (Fig. 7A). In cAF, however, the kinase inhibitors did not modulate CCh-activated IK,ACh, and correspondingly, there was no change in RMP (Fig. 7A,B).


Figure 7
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  		Fig. 7 Effects of kinase inhibitors on CCh-activated current in SR and cAF. A, Effects of PKG, PKC, CaMKII and PKA inhibitors (KT5823, BIM-I, KN-93 and KT5720, respectively) on S2/S1 ratio of Peak- and QSS-IK,ACh in both groups. B, Effects of kinase inhibitors on CCh-induced changes in RMP in both group. Numbers indicate myocytes/patients. *P<0.05 vs. control in SR and cAF. #P<0.05 vs. negative controls.

 
3.5 Protein levels of PKC isoforms
Levels of proteins were normalized to the tissue amount of calsequestrin, which was similar in SR and cAF (Fig. 8). The protein level of PKC{varepsilon}, but not PKC{alpha}, PKCβ1 or PKC{delta}, was ~40% higher in cAF than in SR (Fig. 8). Compared to SR the cAF patients had larger left atria and more frequently valvular heart disease but comparable other clinical parameters and medications (Supplementary data). There was no significant interaction effect between left atrial diameter or presence of valvular heart disease and cAF on PKC{varepsilon} (not shown). Thus, abnormal PKC activity and specifically PKC{varepsilon} is likely to contribute to constitutive IK,ACh activity.


Figure 8
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  		Fig. 8 Representative immunoblots and densitometric analysis of PKC isoforms in SR and cAF. Numbers indicate hearts; CSQ = calsequestrin; *P<0.05 vs. SR.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 Acknowledgments
 References
 
Evidence for abnormal phosphorylation-dependent regulation of constitutively active and CCh-activated IK,ACh is provided by several observations: (i) basal current in SR, consisting of IK1 only, was not affected by inhibition of protein phosphatases, PKC or CaMKII, whereas in cAF it decreased after inhibition of PKC (but not of CaMKII). (ii) The increased protein abundance of PKC{varepsilon} is likely to contribute to the promotion of constitutive IK,ACh activity by PKC; (iii) selective inhibition of PP1 and PP2A reduced CCh-activated Peak- and QSS-IK,ACh in SR but not in cAF suggesting impaired GRK function; (iv) the selective block of PKG, PKC or CaMKII decreased the CCh-activated Peak-IK,ACh without affecting QSS-IK,ACh only in SR but not in cAF. Our results indicate that the occurrence of constitutive IK,ACh activity in cAF may result from abnormal IK,ACh channel phosphorylation by PKC, whereas the additional muscarinic receptor-mediated IK,ACh activation apparently occurs without significant contribution of PKG, PKC or CaMKII.

4.1 Comparison with previous studies
We have recently shown that IK,ACh develops constitutive activity in cAF patients [5]. In dogs with pacing-induced tachycardia, atria and pulmonary veins develop a similar IK,ACh-like component of basal current [6,7]. Blockade of these channels with the selective blocker tertiapin prevents induction of AF episodes [6] suggesting that IK,ACh is a major contributor to initiation and perpetuation of AF. However, pAF patients did not possess a tertiapin-sensitive component of basal current indicating that constitutive IK,ACh activity is a hallmark of cAF, but not of pAF, and thus most likely a consequence rather than a cause of the arrhythmia.

Consistent with ~40% reduction in protein levels of the GIRK1 subunit [24] the muscarinic receptor-activated IK,ACh was ~40% smaller in pAF than in SR. The 10-fold lower potency of tertiapin in pAF (present study) than in cAF [5] suggests also impaired channel regulation. Thus modulation of atrial function by vagal excitation appears limited in pAF patients.

The molecular basis of constitutively active IK,ACh is incompletely understood. In cAF open probability of agonist-independent IK,ACh is higher than in SR without concomitant alterations of other current properties [5]. Lack of effect of the muscarinic receptor blocker atropine demonstrates that constitutive IK,ACh activity is an agonist-independent process [5,7]. Increased receptor-independent dissociation of G{alpha}- and Gβ{gamma}-subunits appears an unlike mechanism because neither pertussis toxin nor absence of GTP affected the IK,ACh-like component of basal current in dogs [7]. These findings point to a modified regulation within the macromolecular IK,ACh complex which contains the catalytic subunits of PKA, PKC, and CaMKII, and PP1 and PP2A [10,11]. Thus, the quantitative and/or qualitative composition of this complex may change in cAF leading to abnormal phosphorylation-dependent IK,ACh regulation.

Activation of IK,ACh requires ATP which may modulate the channel through several mechanisms: (i) transphosphorylation between adenosine- and guanosine-nucleosides via nucleoside diphosphate kinase (NDPK) [8]; (ii) generation of phosphatidylinositol-4,5-bisphosphate (PIP2) via hydrolysis of ATP [25] and (iii) direct phosphorylation of the channels and/or their regulators by protein kinases [10,19]. Recent work, however, challenged the NDPK hypothesis as explanation for agonist-independent channel activation [26]. Although the levels of high-energy phosphates remain stable in cAF [27], high membrane levels of PIP2 underlying the constitutive IK,ACh activity cannot be excluded. The phosphorylation state of muscarinic receptors and IK,ACh channels is dynamically regulated by different kinase and phosphatase subtypes. Consistent with results in animals [19] inhibition of PP1/PP2A reduced CCh-activated IK,ACh in SR suggesting that the shift in the kinase/phosphatase balance to higher channel phosphorylation cannot compensate the stronger muscarinic receptor phosphorylation thereby limiting the activation of IK,ACh (Fig. 3). In contrast PP1/PP2A inhibition was without effect on CCh-activated IK,ACh in cAF indicating abnormal function of GRK.

The regulation of muscarinic receptor function does not involve phosphorylation by CaMKII, PKC and PKA (Fig. 3) [22]. This allows to investigate the contribution of these kinases to channel regulation without confounding effects on muscarinic receptor function. The GIRK1 and GIRK4 channel subunits possess phosphorylation sites for PKA, PKC, and CAMKII and possibly PKG [10,11] and regulation of CCh-activated IK,ACh was modulated by PKC, CaMKII and PKG but not by PKA. In SR, the kinase inhibitors reduced CCh-activated Peak-IK,ACh without an effect on QSS-IK,ACh. Since Peak-IK,ACh reflects the direct binding of the Gβ{gamma}-subunits to the channel and the strength of this binding depends on the actual degree of channel phosphorylation (Fig. 3) [10], PKG-, PKC- and CaMKII-mediated channel phosphorylation may stabilize the binding of Gβ{gamma}-subunits to the channel increasing IK,ACh. In contrast, the kinase inhibitors were without effect on CCh-activated IK,ACh in cAF. This was unexpected, because the protein levels of CaMKII{delta} and PKC{varepsilon} are higher in cAF than in SR [28]. Also, we consistently found that the activity of PP1 and PP2A is higher in cAF than in SR and does not translate into homogeneous changes of protein phosphorylation [13,14]. Thus the lack of contribution of these kinases to CCh-activated IK,ACh in cAF may result from a stronger channel dephosphorylation (Fig. 3). Alternatively, the channels may be hyperphosphorylated due to abnormal control of kinase/phosphatase signaling in the macromolecular complex [14] and inhibition of a single kinase may not reduce the channel's phosphorylation state below the threshold required to impair CCh-activated IK,ACh. Further work is needed to verify these hypotheses.

Inhibition of PP1/PP2A and calcineurin, and several protein kinases (PKA, PKC and CaMKII) did not affect basal current in SR. Phosphorylation of IK1 channels by protein kinases results in current inhibition [29,30] and the PKG-inhibitor KT5823 reduced basal current in SR and cAF. This inhibition, however, was of similar magnitude in both groups suggesting potential block of IK1 channels. In the kidney, PKG increases the amplitude of inward rectifier K+ current [31] pointing to the involvement of PKG in IK1 regulation. Further studies are required to clarify the role of PKG in IK1 regulation.

The CaMKII-inhibitor KN-93 had no effect on basal current in cAF rendering the contribution of CaMKII to either IK1 or constitutively active IK,ACh channels unlikely. Surprisingly, inhibition of PKC with BIM-I reduced basal current in cAF by ~3.5 pA/pF. In human atria activation of G{alpha}q-coupled receptors results in translocation of PKC{delta} and PKC{varepsilon} (but not PKC{alpha} or PKCβ) to the membrane [32], and the protein levels of PKC{varepsilon} are higher in cAF than in SR. Thus, the inhibition of basal current by BIM-I in cAF may result from abnormal contribution of PKC{varepsilon}. Since BIM-I was not effective on basal current in SR (only IK1 present) and activation of PKC inhibits human IK1 [33], the BIM-I-associated decrease in basal current in cAF must involve impaired regulation of constitutively active IK,ACh. Thus while development of constitutively active IK,ACh in cAF requires abnormal channel phosphorylation by PKC, the additional muscarinic receptor-mediated IK,ACh activation apparently does not need the contribution of PKG, PKC or CaMKII.

4.2 Study limitations
Our results are not consistent with data from expression systems and neonatal myocytes where the increase in PKC activity inhibits IK,ACh [11,34]. The reason for this inconsistent observation is unknown, though artificial systems lack important endogenous regulators, and the composition of the IK,ACh channel differs between neonatal and adult myocytes [35]. The predominant subunit in neonatal rat myocytes is GIRK4 [35], whereas in diseased human atria GIRK1 predominates (unpublished data, 2002). Thus, species differences and the concomitant cardiac disease and/or patients' medication could also contribute to the discrepant findings.

We studied the contribution of serine/threonine kinases only. However, mitogen-activated protein kinases (MAPKs) may also contribute to the regulation of IK,ACh. PKC{varepsilon} is known to activate ERK1/2 [36,37] and may stimulate JNK and p38 [37] through an indirect increase of PKC{delta} [38]. Thus, we cannot exclude that MAPKs regulate IK,ACh and that the effects of PKC on constitutively active IK,ACh are mediated by stimulation of MAPKs. An inactive analog of KT5823 was not available to us. Thus, our results with this drug might be confounded by non-specific effects on IK1.

4.3 Clinical implications
In vivo, constitutively active IK,ACh is expected to shorten action potential duration, to enhance atrial vulnerability to tachyarrhythmia and to sustain AF [1]. Here we demonstrate that the molecular basis of constitutively active IK,ACh in cAF may involve abnormal phosphorylation-dependent regulation by PKC. To the best of our knowledge this is the first demonstration of increased PKC{varepsilon} expression in cAF. This may contribute to contractile dysfunction [39,40], hypertrophy and fibrosis, however, proof of these hypotheses warrants further investigations.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
Acknowledgments
 References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.02.009.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
References
 
The authors thank Manja Schöne, Trautlinde Thurm and Ulrike Heinrich for excellent technical assistance.

These studies were supported by the Deutsche Forschungsgemeinschaft (Do 769/1-1,2 to D.D.).


   Notes
 
Time for primary review 31 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 Acknowledgments
 References
 

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