Correspondence to: Wolfgang Schreibmayer, Graz University, Harrachgasse 21/4, A-8010 Graz, Austria. Fax:43 316 380 9660 E-mail:wolfgang.schreibmayer{at}kfunigraz.ac.at.
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Abstract |
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To investigate possible effects of adrenergic stimulation on G proteinactivated inwardly rectifying K+ channels (GIRK), acetylcholine (ACh)-evoked K+ current, IKACh, was recorded from adult rat atrial cardiomyocytes using the whole cell patch clamp method and a fast perfusion system. The rise time of IKACh was 0.4 ± 0.1 s. When isoproterenol (Iso) was applied simultaneously with ACh, an additional slow component (11.4 ± 3.0 s) appeared, and the amplitude of the elicited IKACh was increased by 22.9 ± 5.4%. Both the slow component of activation and the current increase caused by Iso were abolished by preincubation in 50 µM H89 {N-[2-((p -bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, a potent inhibitor of PKA}. This heterologous facilitation of GIRK current by ß-adrenergic stimulation was further studied in Xenopus laevis oocytes coexpressing ß2-adrenergic receptors, m2 -receptors, and GIRK1/GIRK4 subunits. Both Iso and ACh elicited GIRK currents in these oocytes. Furthermore, Iso facilitated ACh currents in a way, similar to atrial cells. Cytosolic injection of 3060 pmol cAMP, but not of Rp-cAMPS (a cAMP analogue that is inhibitory to PKA) mimicked the ß2-adrenergic effect. The possibility that the potentiation of GIRK currents was a result of the phosphorylation of the ß-adrenergic receptor (ß2AR) by PKA was excluded by using a mutant ß2AR in which the residues for PKA-mediated modulation were mutated. Overexpression of the subunit of G proteins (G
s) led to an increase in basal as well as agonist-induced GIRK1/GIRK4 currents (inhibited by H89). At higher levels of expressed G
s, GIRK currents were inhibited, presumably due to sequestration of the ß/
subunit dimer of G protein. GIRK1/GIRK5, GIRK1/GIRK2, and homomeric GIRK2 channels were also regulated by cAMP injections. Mutant GIRK1/GIRK4 channels in which the 40 COOH-terminal amino acids (which contain a strong PKA phosphorylation consensus site) were deleted were also modulated by cAMP injections. Hence, the structural determinant responsible is not located within this region. We conclude that, both in atrial myocytes and in Xenopus oocytes, ß-adrenergic stimulation potentiates the ACh-evoked GIRK channels via a pathway that involves PKA-catalyzed phosphorylation downstream from ß2AR.
Key Words: G proteinactivated inwardly rectifying K+ channels , protein kinase A, heterologous facilitation, cardiomyocytes, Xenopus
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INTRODUCTION |
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Adaptation of cardiac output by the sympathetic nerve acts through potentiation of voltage-dependent Ca2+ and Na+ channel function resulting from the activation of the stimulatory subunit of G proteins (G
s),1 adenylyl cyclase, and subsequent phosphorylation of these channels by cAMP-dependent protein kinase (PKA;
i inhibits adenylyl cyclase and subsequently PKA, while direct activation of G-proteinactivated inwardly rectifying K+ channels (GIRK) is achieved by simultaneous release of the ß/
subunit dimer of G protein (Gß
) (for review, see
S-activated G
s attenuates G ß
-induced activation of GIRK channels expressed in Xenopus oocytes in a membrane-delimited manner (
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MATERIALS AND METHODS |
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Electrophysiology
Atrial cells were enzymatically disaggregated from hearts of adult Sprague-Dawley rats as described (, pulled from standard hematocrit capillaries (564, L/M-3P-A puller; List Electronik). Whole cell current recordings were obtained by rupturing the membrane patch under the tip of the glass pipette by suction. Pipette solutions for perforated patch recordings were made by mixing 100 µl of a nystatin stock solution (50 mg/ml nystatin dissolved in DMSO in the original solution) with 10 ml pipette solution, sonicated and filtered through 200-µm ultrafilters before use. The amount of nystatin needed to get electrical perforation within 12 min after gigaseal formation varied within a range of 15x and depended on the atrial cell preparation. Current recordings were obtained by keeping the cell's membrane potential constant at -80 mV and superfusing with HP medium (see below) containing 10-5 M acetylcholine and/or 10-6 M isoproterenol. H89 {N -[2-((p -Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide} was introduced into the cytosol by adding 5 x 10-5 M to the pipette solution. In addition to a regular, gravitation driven slow perfusion with HP, we also used a fast perfusion system (ALA Scientific) that allowed the fast exchange of medium surrounding the myocyte within 1 s. Current traces were Bessel low-pass filtered at 20 Hz and digitized at 50 Hz using the Labmaster-TL-1 interface (Axon Instruments) connected to a standard Pentium PC using Axotape software (Axon Instruments). The traces were analyzed using Fetchan 6.0 (Axon Instruments). For the evaluation of onset kinetics of IKACh, only those cells were selected whose washout kinetics of acetylcholine indicated efficient fast superfusion (i.e., the time course of washout was exponential). The rising phase of I KACh was fitted to standard one- or two-exponential equations using Sigmaplot (Jandel Scientific). Xenopus laevis oocytes were prepared as described (
C40, 0.01250.2 GIRK4wt, 0.01250.0375 GIRK4
C40, 0.0750.75 GIRK2wt, 0.022 G
s, 1.5 Gß2, 1.5 G
1, 10 CFTR. To knock out the endogenously existing GIRK5 subunit, 80 ng/oocyte of the phosphothioated antisense oligonucleotide KHA2 was injected together with the cRNAs (
Molecular Biology
Plasmid vectors were constructed, grown in bacteria, isolated, and linearized using standard procedures (s (
1 (
C40 and GIRK4
C40, we performed a PCR procedure with forward and reverse primers containing the desired parts of the coding sequence of each cDNA preceded or followed, respectively, by restriction enzyme recognition sequences. The PCR products were digested with the restriction enzymes and ligated into the EcoRI and HindIII sites (GIRK1
C40) and Sma1 and XbaI sites (GIRK4
C40) of pGEMHE.
Data Normalization and Statistics
To normalize our data (to eliminate scatter introduced by batch-to-batch variation of protein expression in oocyte preparations, different ratios of basal to acetylcholine induced currents for different GIRK isoform combinations or mutants), all currents were expressed as a percentage of the basal HK-induced current of the GIRK1/GIRK4 heterooligomeric channel of a given experimental day and batch of oocytes. Different groups were compared using the unpaired Student's t test (Sigmaplot 2.0; Jandel Scientific). In some instances, the calculated average value of current increase in percent was tested for a significant difference from zero by assuming a normal distribution (
Solutions
The composition of the solutions used was as follows (mM): HP: 136 KCl, 4 NaCl, 2 MgCl2, 10 HEPES, buffered with NaOH to pH 7.4; pipette solution: 120 K+/aspartate, 20 NaCl, 2 MgCl2 , 11 EGTA, 1 CaCl2, 2 ATP, 0.1 GTP, 10 HEPES, buffered with KOH to pH 7.4; ND96: 96 NaCl, 2 KCl, 1 MgCl2, 1 CaCl 2, 5 HEPES, buffered with NaOH to pH 7.4; NDE: same as ND96, but contained 2.5 mM pyruvate, 0.1% antibiotics (G-1397, 1,000x stock; Sigma Chemical Co.) and 1.8 CaCl2 ; HK: 96 KCl, 2 NaCl, 1 MgCl2, 1 CaCl2, 5 HEPES, buffered with KOH to pH 7.4.
Chemicals
All chemicals used were reagent grade throughout. Reagents for molecular biology were purchased from Boehringer-Mannheim or MBI Fermentas.
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RESULTS |
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IKACh of freshly dissociated rat atrial cells was elicited by superfusion of the cells with 10-5 M acetylcholine in HP solution. The intracellular solution contained 2 mM ATP to allow the production of cAMP. ß2AR coactivation by coapplication of 10-6 M isoproterenol enhanced IKACh by 2025% (Fig 1A and Fig B). Furthermore, the onset kinetics of IKACh were changed: when only acetylcholine was present, the activation kinetics followed a monoexponential function with a time constant in the range of 0.4 s; an additional, slower time constant in the range of 10 s appeared upon isoproterenol coapplication (Fig 1B and Fig C; Table 1). When isoproterenol was applied after the onset of I KACh, an additional increase of IKACh could also be observed, which was comparable in size to the ß-adrenergic effect upon coapplication. This additional increase of IKACh was also observed when the whole-cell recording was performed using the perforated patch technique with nystatin, leaving the macromolecular composition of the cytosol intact (Fig 1 B). Application of 10-6 M isoproterenol alone did not result in any detectable IKACh or increase in inward current (n = 5, data not shown). When H89, a specific inhibitor of PKA, was included in the patch pipette, the ß-adrenergic effects, both on magnitude as well as on kinetics of IKACh, disappeared (Fig 1).
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To investigate the details of the signal transduction pathway leading to the ß-adrenergic effect on IKACh, we used a heterologous expression system, the Xenopus laevis oocyte. To assess the ability of heterologously expressed ß2AR to activate PKA in Xenopus oocytes, we coexpressed ß2AR together with the CFTR, a Cl- channel that is a known target for PKA phosphorylation (s, isoproterenol produced a marked increase in inward current, which was equal in magnitude to CFTR inward currents produced by cAMP injections (Fig 2A and Fig B). Furthermore, isoproterenol-induced CFTR currents were prevented and/or blocked by cytosolic injections of Rp-cAMPS ( Fig 2 A; summarized in C), demonstrating that the isoproterenol response was entirely due to PKA stimulation and that Rp-cAMPS injections are indeed effective in blocking endogenous PKA in Xenopus laevis oocytes.
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Modulation of GIRK channels by ß-adrenergic receptors was studied in Xenopus oocytes heterologously expressing muscarinic m2 -receptor, ß-adrenergic receptors, and two subunits of G-proteinactivated, inwardly rectifying K+ channels (GIRK1 and GIRK4, respectively). Heteromeric GIRK1/GIRK4 channels are believed to constitute the atrial GIRK (see -subunits released from ß-adrenergic receptors can directly activate GIRK channels in Xenopus oocytes and atrial cells, provided that exogenous Gß and/or G
s are overexpressed (
effects and to distinguish them from effects put forth by the cytosolic second messenger branch, 3'5'-cAMP was directly injected into the oocytes during electrophysiological recording. An enhancement of inward current resulted both in the absence and the presence of acetylcholine (Fig 3C and Fig E). The current voltage relation of cAMP-induced currents clearly revealed inward rectification (Fig 3 F). Furthermore, cAMP effects on IHK were absent when cRNA encoding GIRK channels was not injected into the oocytes. The enhancement of the basal activity of GIRK channels by cAMP injection was not altered by ß2AR coexpression [59.6 ± 7.5% (n = 55, see Fig 3 D) vs. 59.7 ± 7.2% (n = 9, data not shown)]. Injection of Rp-cAMPS, a cAMP analogue and inhibitor of PKA, did not enhance the basal inward currents. This fact indicates that nonspecific effects of cyclic nucleotides, unrelated to PKA activation, were absent. To better understand the mechanisms of modulation of GIRK via ß2AR and G
s in Xenopus oocytes and to substantiate the finding that PKA might stimulate basal and agonist-induced GIRK currents in the oocytes, we overexpressed G
s at different concentrations in addition to GIRK1, GIRK4, and m2R. At low doses of injected cRNA encoding G
s, basal and ACh-evoked GIRK currents were enhanced, while at higher doses a suppression of both current components was observed. Interestingly, the increase in both basal and agonist-induced currents was inhibited by preincubation of the oocytes in 5 x 10-5 mol/liter H89, while the suppression at higher expression levels of G
s was H89 insensitive (Fig 4). These results suggest that the basal activity of the overexpressed G
s is sufficient to activate adenylyl cyclase, and thus PKA, and to cause an increase in GIRK activity. At higher doses, the free G
s most likely sequesters Gß
, thus causing a decrease in GIRK activity.
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In the intact atrium, stimulation of GIRK occurs via PTX-sensitive G proteins of the Gi/Go family (reviewed by s to G
i/G
o when ß 2AR is phosphorylated by PKA (
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Phosphorylation of seven-helix receptors is an important mechanism for the regulation of receptor activity (
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To investigate whether other isomeric forms of GIRK channels are also modulated by PKA, we expressed different subunit compositions: GIRK1/GIRK5, GIRK1/GIRK2, and the homooligomeric GIRK2 channels. For combinations not containing GIRK5, the endogenous GIRK5 subunit was eliminated by coinjection of a subunit-specific antisense oligonucleotide ( binding (
binding regions. Cytosolic cAMP injections enhanced currents of mutant channels comprised of both GIRK1
C40/GIRK4
C40 . The cAMP-induced currents had the following magnitude, when normalized to the basal current of the oocytes expressing GIRK1wt /GIRK4wt: cAMP injections on basal currents: GIRK1
C40/GIRK4
C40, 33.3 ± 8.7% (n = 9), compared with 66.1 ± 8.0% (n = 40) for GIRK1 wt/GIRK4wt; cAMP injections during ACh superfusion: GIRK1
C40/GIRK4
C40, 67.2 ± 28.0% (n = 6) compared with 100.1 ± 22.9% (n = 13) for GIRK1wt/GIRK4wt. All cAMP effects deviate significantly from zero at the P < 0.001 level. Hence, the distal COOH terminus (last 40 aminoacids) of the GIRK channel is not the structural determinant that mediated the PKA effect.
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DISCUSSION |
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Our results clearly show that IKACh in native atrial myocytes derived from adult rats is enhanced by ß-adrenergic stimulation. This result strongly supports the finding of effect. Furthermore, in native atrial cells, direct G-protein activation by ß2ARs has been shown to occur only after heterologous overexpression of G
s (
s-induced PKA phosphorylation, but in other preparations, PKA effects on IKACh were absent (
dimers and PKA phosphorylation are required for GIRK activation.
To study the molecular mechanism of PKA-induced GIRK stimulation in more detail, we employed the Xenopus laevis oocyte expression system. First, the CFTR Cl- channels were expressed as reporters of PKA activity, since PKA activation of these channels is well documented (s, is able to activate PKA to the same extent as direct cytosolic injections of the second messenger. Furthermore, cytosolic injections of Rp-cAMPS (a selective inhibitor of PKA) completely abolished ß-adrenergic stimulation of CFTR, demonstrating that PKA activity is completely blocked under these conditions. Further evidence for PKA regulation of GIRK was derived using this system: intracellular injection of cAMP, but not of Rp-cAMPS, stimulated GIRK currents to the same extend as heterologously coexpressed ß2AR (Fig 3). This finding is substantiated by the fact that modest overexpression of G
s leads to an increase of basal (agonist independent) as well as acetylcholine-activated GIRK current that can be inhibited by H89, a selective blocker of PKA (Fig 4). At higher levels of overexpression, however, GIRK activity decreases, most likely by sequestering endogenously available G ß
subunits (see
i /G
o. On the other hand, PTX treatment significantly reduced isoproterenol-induced GIRK currents in the case of ß2 ARwt (but not of ß2ARPF), indicating that coupling of ß2ARwt to G
i/G
o also contributes to some extent. Hence, I Iso in Xenopus laevis oocytes is comprised of direct G-protein effects (both PTX sensitive and insensitive) in addition to the part contributed by PKA.
A strong PKA phosphorylation consensus site has been reported to exist in the very end of the carboxy terminus of the GIRK1 subunit (
In native atrial cells, a heterooligomeric protein comprising the GIRK1 and GIRK4 subunits produces most of IKACh. From our study, it becomes evident that GIRK1/GIRK4 heterooligomeric channels are subject to PKA stimulation in native atrial cells as well as in the heterologous expression system. In addition, we were able to demonstrate that other GIRK subunit combinations are subject to PKA-induced facilitation: GIRK1/GIRK5, GIRK1/GIRK2, and even homooligomeric GIRK2 channels were found to be regulated by PKA in the same manner. This finding indicates that we are dealing with a phenomenon of broad functional significance for GIRK channel physiology. Stimulatory and inhibitory G-proteins are thought to produce opposing effects on cardiac excitability under normal physiological conditions. In the present study, however, this is not the case. The physiological role of such heterologous facilitation could be understood as follows: in the heart, ß-adrenergic stimulation is known to act via voltage-dependent Ca2+ and Na+ channels, thereby promoting depolarization, excitability, and contraction (
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Footnotes |
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Dr. Uezono's present address is Department of Pharmacology, Miyazaki Medical College, School of Medicine, Kiyotake 889-1692, Miyazaki, Japan.
1 Abbreviations used in this paper: ACh, acetylcholine; ß 2AR: ß2-adrenergic receptor; G ,
subunit of G proteins; Gß
, ß/
subunit dimer of G protein; GIRK, G proteinactivated inwardly rectifying K+ channel; HK, high-K+ extracellular medium; Iso, isoproterenol; m2R, muscarinergic acetylcholine receptor subtype 2; PKA, cAMP- activated protein kinase; PTX, pertussis toxin.
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Acknowledgements |
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Ms. C. Lorenz (Graz, Austria) provided excellent technical support. The authors thank K. Groschner (Graz, Austria), A. Kovoor (Pasadena, CA), and L. Weigl (Vienna, Austria) for critically reading the manuscript. We thank Y. Daaka and R. Lefkowitz for the cDNA of ß2ARPF .
This study was supported by the Austrian Science Foundation (SFB708, P11560-MED, P13724-GEN), the Austrian National Bank (OENB7716), the National Institutes of Health (GM56260), and the Israel Basic Research Fund.
Note added in proof: During the review process of this paper, Medina et al. reported at the 1999 APS conference, "Biology of Potassium Channels: From Molecules to Disease," that the GIRK1 subunit itself is phosphorylated in vitro by PKA (Medina, I., G. Krapivinsky, P. Kovoor, L. Krapivinsky, and D.E. Clapham. 1999. Channel phosphorylation is required for IKACh activation by G ß. Physiologist. 42:A-17).
Submitted: 25 August 1999
Revised: 6 March 2000
Accepted: 7 March 2000
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