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• W1989821836 abstract "G protein-activated inwardly rectifying K+ (GIRK) channels, expressed in atrial myocytes, various neurons, and endocrine cells, represent the paradigmatic target of βγ subunits released from activated heterotrimeric G proteins. These channels contribute to physiological slowing of cardiac frequency and synaptic inhibition. They are activated by βγ dimers released upon stimulation of receptors coupled to pertussis toxin-sensitive G proteins (Gi/o), whereas βγ released from Gs do not converge on the channel subunits. This is in conflict with the finding that dimeric combinations of various β and γ subunits can activate GIRK channels with little specificity. In the present study, we have overexpressed the major subtypes of cardiac β-adrenergic receptors (β1-AR and β2-AR) in atrial myocytes by transient transfection. Whereas in native cells β-adrenergic stimulation with isoproterenol failed to induce measurable GIRK current, robust currents were recorded from myocytes overexpressing either β1-AR or β2-AR. Whereas the β2-AR-induced current showed the same sensitivity to pertussis toxin as the current evoked by the endogenous Gi/o-coupled muscarinic M2 receptor, isoproterenol-activated currents were insensitive to pertussis toxin treatment in β1-AR-overexpressing myocytes. In contrast to a recent publication (Leaney, J. L., Milligan, G., and Tinker, A. (2000) J. Biol. Chem. 275, 921–929), sizable GIRK currents could also be activated by isoproterenol when the signaling pathway was reconstituted by transient transfection in two different standard cell lines (Chinese hamster ovary and HEK293). These results demonstrate that specificity of receptor-G protein signaling can be disrupted by overexpression of receptors. Moreover, the α subunit of heterotrimeric G proteins does not confer specificity to Gβγ-mediated signaling. G protein-activated inwardly rectifying K+ (GIRK) channels, expressed in atrial myocytes, various neurons, and endocrine cells, represent the paradigmatic target of βγ subunits released from activated heterotrimeric G proteins. These channels contribute to physiological slowing of cardiac frequency and synaptic inhibition. They are activated by βγ dimers released upon stimulation of receptors coupled to pertussis toxin-sensitive G proteins (Gi/o), whereas βγ released from Gs do not converge on the channel subunits. This is in conflict with the finding that dimeric combinations of various β and γ subunits can activate GIRK channels with little specificity. In the present study, we have overexpressed the major subtypes of cardiac β-adrenergic receptors (β1-AR and β2-AR) in atrial myocytes by transient transfection. Whereas in native cells β-adrenergic stimulation with isoproterenol failed to induce measurable GIRK current, robust currents were recorded from myocytes overexpressing either β1-AR or β2-AR. Whereas the β2-AR-induced current showed the same sensitivity to pertussis toxin as the current evoked by the endogenous Gi/o-coupled muscarinic M2 receptor, isoproterenol-activated currents were insensitive to pertussis toxin treatment in β1-AR-overexpressing myocytes. In contrast to a recent publication (Leaney, J. L., Milligan, G., and Tinker, A. (2000) J. Biol. Chem. 275, 921–929), sizable GIRK currents could also be activated by isoproterenol when the signaling pathway was reconstituted by transient transfection in two different standard cell lines (Chinese hamster ovary and HEK293). These results demonstrate that specificity of receptor-G protein signaling can be disrupted by overexpression of receptors. Moreover, the α subunit of heterotrimeric G proteins does not confer specificity to Gβγ-mediated signaling. β-adrenergic receptor β1- and β2-adrenergic receptor subtypes G protein-gated inward rectifier K+ acetylcholine muscarinic M2 receptor pertussis toxin G protein-coupled receptor kinase 2 isoproterenol atrial GIRK current human embryonic kidney Chinese hamster ovary enhanced green fluorescent protein cAMP-dependent protein kinase The β-adrenergic receptors (β-AR)1 belong to the superfamily of G protein-coupled receptors. The classical view of β-AR signaling is that the agonist-activated receptor interacts with the stimulatory type of G protein (Gs), which, via its α subunit, activates adenylate cyclase, resulting in phosphorylation of target proteins by protein kinase A (PKA), such as thel-type Ca2+ channel and phospholamban. In the heart β-ARs play an important role in acute modulation of cardiac output in response to variable demands of the organism by increasing pacemaker frequency and myocardial contractility. Apart from this classic role in acute regulation of mechanical and electrical functions of the heart, β-AR may be involved in long term control of myocytes including cell survival and apoptosis under various conditions (1Chesley A. Lundberg M.S. Asai T. Xiao R.P. Ohtani S. Lakatta E.G. Crow M.T. Circ. Res. 2000; 87: 1172-1179Crossref PubMed Scopus (338) Google Scholar,2Zhu W.Z. Zheng M. Koch W.J. Lefkowitz R.J. Kobilka B.K. Xiao R.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1607-1612Crossref PubMed Scopus (371) Google Scholar). The predominant type of cardiac β-AR is the β1-AR. There is, however, general agreement that β2-AR are functionally expressed in ventricular and atrial myocytes of various species including man (3Brodde O.-E. Michel M.C. Pharmacol. Rev. 1999; 51: 651-689PubMed Google Scholar, 4Post S.R. Hammond H.K. Insel P.A. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 343-360Crossref PubMed Scopus (133) Google Scholar). Until several years ago, it had been almost a dogma that β-adrenergic signal transduction in cardiac cells proceeds exclusively via the stimulatory G protein (Gs), resulting in activation the cAMP/PKA-dependent signaling pathway (5Xiao R.-P. Cheng H. Zhou Y.-Y. Kuschel M. Lakatta E.G. Circ. Res. 1999; 85: 1092-1100Crossref PubMed Scopus (218) Google Scholar). Whereas this seems to be uncontradicted for the β1-AR, there is a growing body of evidence that β2-AR couples to both Gs and Gi/o(5Xiao R.-P. Cheng H. Zhou Y.-Y. Kuschel M. Lakatta E.G. Circ. Res. 1999; 85: 1092-1100Crossref PubMed Scopus (218) Google Scholar, 6Communal C. Singh K. Sawyer D.B. Colucci W.S. Circulation. 1999; 100: 2210-2212Crossref PubMed Scopus (497) Google Scholar, 7Xiao R.-P. Avdonin P. Zhou Y.-Y. Cheng H. Akhter S.A. Eschenhagen T. Lefkowitz R.J. Koch W.J. Lakatta E.G. Circ. Res. 1999; 84: 43-52Crossref PubMed Scopus (329) Google Scholar, 8Kuschel M. Zhou Y.-Y. Cheng H. Zhang S.-J. Chen Y. Lakatta E.G. Xiao R.-P. J. Biol. Chem. 1999; 274: 22048-22052Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 9Kilts J.D. Gerhardt M.A. Richardson M.D. Sreeram G. Mackensen G.B. Grocott H.P. White W.D. Davis R.D. Newman M.F. Reves J.G. Schwinn D.A. Kwatra M.M. Circ. Res. 2000; 87: 705-709Crossref PubMed Scopus (129) Google Scholar). Additional interesting differences in signaling between β1-AR and β2-AR may also arise from differences in subcellular localization and organization with other signaling components (8Kuschel M. Zhou Y.-Y. Cheng H. Zhang S.-J. Chen Y. Lakatta E.G. Xiao R.-P. J. Biol. Chem. 1999; 274: 22048-22052Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 10Jurevicius J. Fischmeister R. Proc. Natl. Acad. Sci. U. S. A. 1996; 96: 295-299Crossref Scopus (307) Google Scholar). Atrial and, to a lesser extent, ventricular myocytes (11Koumi S.-I. Wasserstrom J.A. Am. J. Physiol. 1994; 266: H1812-H1821PubMed Google Scholar, 12Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (854) Google Scholar) express the prototype of a G protein-gated inwardly rectifying K+ channel (K(ACh)). This channel is composed, in a heterotetrameric fashion of GIRK1 channel (G protein-coupled inwardly rectifying K+ channel 1) and GIRK4 channel (also termed Kir3.1 and Kir3.4 subunits). In the heart K(ACh) channels contribute to mediating the negative chronotropic effect of vagally released acetylcholine (ACh) via muscarinic M2 receptors (13Wickman K. Nemec J. Gendler S.J. Clapham D.E. Neuron. 1998; 20: 103-114Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) (M2ACh-R). Similar channels with different subunit composition are expressed in various endocrine cells and in the central nervous system, where they are supposed to be involved in inhibitory synaptic mechanisms (see Refs. 14Yamada M. Inanobe A. Kurachi Y. Pharmacol. Rev. 1998; 50: 723-757PubMed Google Scholar, 15Karschin A. News Physiol. Sci. 1999; 14: 215-220PubMed Google Scholar, 16Mark M.D. Herlitze S. Eur. J. Biochem. 2000; 267: 5830-5836Crossref PubMed Scopus (192) Google Scholar for reviews). These channels have in common that their gating is controlled by direct interaction of their subunits with βγ subunits of heterotrimeric G proteins (12Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (854) Google Scholar,17Perez-Terzic C. Pyle J. Jaconi M. Clapham D.E. Stehno-Bittel L. Science. 1996; 273: 1875-1877Crossref PubMed Scopus (161) Google Scholar). In native atrial myocytes, only receptors coupled to pertussis toxin (Ptx)-sensitive G proteins (Gi/Go) were believed to couple to K(ACh) channels, such as M2 muscarinic, A1 purinergic, and a sphingolipid receptor belonging to the EDG family (18Bünemann M. Liliom K. Brandts B. Pott L. Tseng J.-L. Desiderio G.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5527-5534Crossref PubMed Scopus (119) Google Scholar, 19Liliom K. Sun G. Bünemann M. Virág T. Nusser N. Baker D.L. Eickel A. Brandts B. Bender K. Wang D. Fabian M.J. Malik K.F. Miller D.D. Desiderio D.M. Tigyi G. Pott L. Biochem. J. 2001; 355: 189-197Crossref PubMed Scopus (144) Google Scholar). More recently, weak activation by stimulation of β-adrenergic receptors has been described in dog atrial myocytes, which could be enhanced by adenoviral infection, a maneuver that is assumed to increase expression of the Gαs subunit (20Sorota S. Rybina R. Yamamoto A. Du X.-Y. J. Physiol.(Lond.). 1999; 514: 413-423Crossref Scopus (22) Google Scholar). Moreover, Mullner et al. (21Mullner C. Vorobiov D. Bera A.K. Uezono Y. Yakubovich D. Frohnwieser-Steinecker B. Dascal N. Schreibmayer W. J. Gen. Physiol. 2000; 115: 547-558Crossref PubMed Scopus (50) Google Scholar) described a facilitation of M2AChR-induced activation of atrial GIRK current (IK(ACh)) in dog atrial myocytes as well as of GIRK channels expressed in Xenopusoocytes, which was suggested to be mediated by PKA phosphorylation at some point downstream of the receptor. These findings contrast with studies from other laboratories (22Wang Y.G. Lipsius S.L. Circ. Res. 1995; 77: 565-574Crossref PubMed Google Scholar, 23Bender K. Wellner-Kienitz M.-C. Meyer T. Pott L. FEBS Lett. 1998; 439: 115-120Crossref PubMed Scopus (10) Google Scholar, 24Wellner-Kienitz M.-C. Bender K. Brandts B. Meyer T. Pott L. FEBS Lett. 1999; 451: 279-283Crossref PubMed Scopus (17) Google Scholar). In those studies no effect of β-adrenergic stimulation on IK(ACh) was found in atrial myocytes under normal conditions. However, manipulation of Gβγ binding to other proteins, such as G protein-coupled receptor kinase 2 (GRK2), in atrial myocytes resulted in a weak but significant activation of IK(ACh) by the unspecific β-agonist isoproterenol (Iso). As compared with the current that could be activated by a saturating concentration of ACh, currents caused by β-adrenergic stimulation in those studies were small. A small current remained following pre-treatment of myocytes with Ptx, using a protocol that completely inhibited the M2 pathway, suggesting that at least part of the β-adrenergic K(ACh) activation was via Gs. Thus, as a rule, no activation of native IK(ACh) is observed upon β-adrenergic stimulation, i.e. by βγ subunits released from Ptx-insensitive Gs. This contrasts with the finding that various βγ combinations can activate K(ACh) channels with little specificity when applied to the internal face of excised membrane patches (25Wickman K.D. Iñiguez-Lluhi J.A. Davenport P.A. Taussig R. Krapivinsky G.B. Linder M.E. Gilman A.G. Clapham D.E. Nature. 1994; 368: 255-257Crossref PubMed Scopus (377) Google Scholar). Moreover, in the oocyte expression system, robust macroscopic GIRK currents can be activated via co-expressed β2 adrenergic receptors (26Lim N.F. Dascal N. Labarga C. Davidson N. Lester H.A. J. Gen. Physiol. 1995; 105: 421-439Crossref PubMed Scopus (90) Google Scholar). To obtain further information on target specificity/promiscuity of β1-AR and β2-AR in their native environment, in the present study these receptors have been overexpressed in adult rat atrial myocytes by transient transfection. In β1-AR- and β2-AR-overexpressing myocytes, isoproterenol induced currents amounting to 70% of maximum IK(ACh). Evaluation of G protein specificity using Ptx showed a predominant contribution of Gi/Go for the β2-AR and a predominant, if not exclusive, contribution of Gs for the β1-AR. These data demonstrate that acutely overexpressed β-AR in atrial myocytes couple promiscuously to a classic target of Gi-derived βγ subunits. As overexpression of β-AR by gene transfer is considered as a therapeutic strategy to treat heart failure (e.g. Refs. 27Maurice J.P. Hata J.A. Shah A.S. White D.C. McDonald P.H. Dolber P.C. Wilson K.H. Lefkowitz R.J. Glower D.D. Koch W.J. J. Clin. Invest. 1999; 104: 21-29Crossref PubMed Scopus (192) Google Scholarand 28Shah A.S. Lilly E. Kypson A.P. Tai O. Hata J.A. Pippen A. Silvestry S.C. Lefkowitz R.J. Circulation. 2000; 101: 408-414Crossref PubMed Scopus (112) Google Scholar), this loss in target specificity should be taken into consideration. Experiments were performed with local ethics committee approval. Wistar Kyoto rats of either sex (around 200 g) were anesthetized by intravenous injection of urethan (1 g/kg). The chest was opened, and the heart was removed and mounted on the cannula of a sterile Langendorff apparatus for coronary perfusion at constant flow. The method of enzymatic isolation of atrial myocytes has been described elsewhere (e.g. Ref. 29Bünemann M. Brandts B. Pott L. J. Physiol.(Lond.). 1996; 492: 351-362Crossref Scopus (42) Google Scholar). The culture medium was fetal calf serum-free bicarbonate-buffered M199 (Life Technologies, Inc., Dreieich, Germany) containing gentamycin (25 μg/ml, Sigma, Deisenhofen, Germany) and kanamycin (25 μg/ml, Sigma). Cells were plated at a low density (several thousand cells per dish) on 36-mm culture dishes. Medium was changed 24 h after plating and then every 2nd day. Myocytes were used experimentally from day 0 until day 5 after isolation. No effects of time in culture were found as for the key experiments. For the patch clamp measurements of IK(ACh), an extracellular solution of the following composition was used (mm): 120 NaCl, 20 KCl, 0.5 CaCl2, 1.0 MgCl2, 10.0 Hepes/NaOH, pH 7.4. The solution for filling the patch clamp pipettes for whole cell measurements of G protein-activated K+ currents contained (mm): 110 potassium aspartate, 20 KCl, 5.0 NaCl, 1.0 MgCl2, 2.0 Na2ATP, 2.0 EGTA, 0.01 GTP, 10.0 HEPES/KOH, pH 7.4. For measurement of l-type Ca2+ current, the pipette filling solution had the following composition (mm): 140 CsCl, 7 MgCl2, 5 Na2ATP, 10 EGTA, 20 HEPES/CsOH, pH 7.3; and 20 μm GTP. Standard chemicals were from Merck (Darmstadt, Germany). EGTA, Hepes, MgATP, Ado, GTP, ACh-chloride, and isoprenaline chloride were from Sigma. CGP 207112A was kindly provided by Novartis Pharma (Basel, Switzerland). Membrane currents were measured using whole-cell patch clamp. Pipettes were fabricated from borosilicate glass and were filled with the solution listed above (DC resistance = 4–6 megohms). Currents were measured by means of a patch clamp amplifier (List LM/EPC 7, Darmstadt, Germany). Signals were analog-filtered (corner frequency of 1–3 kHz), digitally sampled at 5 kHz, and stored on a computer, equipped with a hardware/software package (ISO2 by MFK, Frankfurt/Main, Germany) for voltage control and data acquisition. Experiments were performed at ambient temperature (22–24 °C). If not otherwise stated, cells were voltage-clamped at −90 mV, i.e. negative to EK, resulting in inward K+ currents. Current-voltage relations were determined by means of voltage ramps between −120 mV and +60 mV. Rapid superfusion of the cells for application and withdrawal of different solutions was performed by means of a solenoid-operated flow system that permitted switching between up to six different solutions (t½ ≤ 100 ms). Myocytes, human embryonic kidney (HEK293) cells, and Chinese hamster ovary (CHO) cells were transfected using the LipofectAMINE (Life Technologies, Inc.) methodology. Myocytes were cultured overnight to allow attachment. For transfection 2.5 μg/plate of the reporter IRES-EGFP receptor (CLONTECH) and 2.5 μg/plate of the constructs pcDNA3.1-β1R (rat, kindly provided by Dr. C. A. Machida) or human pcDNA-β2R, respectively, were used. All cDNAs were under cytomegalovirus promotor control. β1-AR cDNA was subcloned into pcDNA3.1 vector using EcoRI and XbaI restriction sites. To precomplex the DNA for each plate, constructs were incubated with 100 μl of transfection medium (M199 without fetal calf serum and antibiotics) and 5 μl of Plus reagent (Life Technologies, Inc.) for 15 min at room temperature. After incubation 2 μl of LipofectAMINE (Life Technologies, Inc.) was diluted in 100 μl of transfection medium, mixed with Plus/DNA solution, and incubated in the same way before dilution of transfection medium to a final volume of 1 ml. Myocytes were washed with prewarmed phosphate-buffered saline and incubated for 3 h under normal cell culture conditions with the transfection solution. Thereafter, dishes were washed with phosphate-buffered saline and incubated with medium M199 supplemented with gentamycin and kanamycin (20 μg/ml each) at 37 °C and 5% CO2. HEK293 cells and CHO cells were transfected using the same protocol but different amounts of DNA. For transfection of both cell lines, 0.5 μg of IRES-EGFP vector, 0.5 μg of pcDNA3-β1R, 0.5 μg of pcDNA3-GIRK1 (mouse), 0.5 μg of pcDNA3.1-GIRK4 was used and incubated as described. As shown previously, in rat atrial myocytes studied under the experimental condition of the present study Iso neither induced a measurable IK(ACh), nor were any of the properties of the current modified. A representative recording of membrane current from a cell transfected with the EGFP vector only is shown in Fig.1. Iso at 10 μm, a concentration that is saturating for both β-AR subtypes, did not cause activation of IK(ACh), nor did the β-agonist affect the response to ACh when applied before or simultaneously with the muscarinic agonist. This behavior is representative of more than 100 cells studied, including freshly isolated cells, non-transfected myocytes cultured for 3–5 days and EGFP-transfected cells cultured for 3–4 days after transfection. Moreover, this was independent of the concentration of ACh ([ACh] ≥ 5*10−8 m ≥ 2*10−5 m). This finding is not limited to myocytes from rats but could be confirmed also in freshly isolated and cultured guinea pig atrial myocytes (data not shown). In none of these cells did exposure to Iso result in activation of IK(ACh)or modification of its properties. This confirms previous studies (23Bender K. Wellner-Kienitz M.-C. Meyer T. Pott L. FEBS Lett. 1998; 439: 115-120Crossref PubMed Scopus (10) Google Scholar,24Wellner-Kienitz M.-C. Bender K. Brandts B. Meyer T. Pott L. FEBS Lett. 1999; 451: 279-283Crossref PubMed Scopus (17) Google Scholar) and clearly demonstrates that in rat and guinea pig atrial myocytes endogenous β-AR fail to activate IK(ACh) by βγ subunits released from the coupling G protein(s). Furthermore, in the system under study, PKA-dependent phosphorylation of the channel subunits or any other component relevant to this signaling pathway does not seem affect the properties of the native current (see also Fig. 5).Figure 5Forskolin fails to activate IK( ACh ) but potentiates L-type Ca2+current. A, representative current recording from a β2-AR-overexpressing myocyte. B, sample traces of l-type Ca2+ currents (leak subtracted) evoked by stepping from −50 mV holding potential to −5 mV; thenumbers refer to panel C, which represents a plot of ICa amplitude versustime.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In EGFP-positive myocytes, co-transfected with the pcDNA-β1 vector, robust Iso-induced currents were recorded. A representative example is illustrated in Fig.2A. In this measurement atropine (1 μm) was superfused before and simultaneously with Iso in order to completely shut off the M2AChR-linked pathway. A comparable result was obtained in myocytes overexpressing the β2-AR subtype (Fig. 2B). Identity of the Iso-induced current with IK(ACh) was confirmed by its current-voltage relation (panel C), which perfectly matched the I/V curve of ACh-induced current. The summarized data from myocytes overexpressing the β1-AR (n = 12) or the β2-AR (n= 12) yielded mean currents of comparable densities for both receptors amounting to ∼70% of IK(ACh) induced by M2AChR stimulation by a saturating concentration of ACh. Atrial IK(ACh) undergoes a partial rapid desensitization, which, as a distinct kinetic component, is only seen upon fast application of agonist, i.e. fast activation of the current (compare Fig. 1). Rapid or acute desensitization can also be identified as a reduction in total current, when two activating agonists are present simultaneously (e.g. Refs. 30Wellner-Kienitz M.-C. Bender K. Meyer T. Bünemann M. Pott L. Circ. Res. 2000; 86: 643-648Crossref PubMed Scopus (29) Google Scholar, 31Kurachi Y. Nakajima T. Sugimoto T. Pflügers Arch. 1987; 410: 227-233Crossref PubMed Scopus (69) Google Scholar, 32Meyer T. Wellner-Kienitz M.-C. Biewald A. Bender K. Eickel A. Pott L. J. Biol. Chem. 2001; 276: 5650-5658Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The molecular nature of this variable component of desensitization is not known yet. It is supposed, however, to reflect a phenomenon related to the channel complex, rather than a receptor desensitization, common to many, if not all, members of the seven-helix superfamily (see Refs. 33Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (853) Google Scholar and 34Bünemann M. Lee K.B. Pals-Rylaarsdam R. Roseberry A.G. Hosey M.M. Annu. Rev. Physiol. 1999; 61: 169-192Crossref PubMed Scopus (133) Google Scholar for reviews). This view is mainly supported by the observation that it is heterologous and it is the more pronounced, the larger the current density, independent of the species of receptor used for activation (e.g. M2AChR, A1-adenosine receptor; Refs. 30Wellner-Kienitz M.-C. Bender K. Meyer T. Bünemann M. Pott L. Circ. Res. 2000; 86: 643-648Crossref PubMed Scopus (29) Google Scholar and 35Bünemann M. Pott L. J. Physiol.(Lond.). 1995; 482: 81-92Crossref Scopus (33) Google Scholar). Moreover, recent evidence suggests that this property of macroscopic IK(ACh) is associated with the GIRK1 subunit, since currents carried by homomeric GIRK4 channels lack a distinct desensitizing component (36Bender K. Wellner-Kienitz M.-C. Inanobe A. Meyer T. Kurachi Y. Pott L. J. Biol. Chem. 2001; 276: 28873-28880Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The heterologous nature of fast desensitization, i.e. its independence on receptor species, is confirmed by the representative current traces from a β2-AR-overexpressing cell presented in Fig. 3. It shows that sensitivity to ACh was substantially reduced on the background of an Iso-activated current, which amounted to 65% of peak ACh-induced IK(ACh). The total current in the presence of both agonists was 74% of peak IK(ACh). As shown previously (e.g. Refs. 30Wellner-Kienitz M.-C. Bender K. Meyer T. Bünemann M. Pott L. Circ. Res. 2000; 86: 643-648Crossref PubMed Scopus (29) Google Scholar and 36Bender K. Wellner-Kienitz M.-C. Inanobe A. Meyer T. Kurachi Y. Pott L. J. Biol. Chem. 2001; 276: 28873-28880Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), this occlusive behavior is due to the fact that desensitization of GIRK channels by Iso took place during the activation phase, resulting in a current trajectory that lacks a distinct desensitizing component. In the lower panel the activation phases of the current induced by rapid application of ACh, Iso, or both agonists simultaneously have been superimposed. Interestingly, not only were the amplitudes of currents induced by ACh or ACh plus Iso, respectively, identical, their activation kinetics were also identical, i.e. activation rate was saturated at 20 μm ACh. Taken together, these data, which qualitatively were confirmed in 9 myocytes (4 β1-AR/5 β2-AR) to which such an experimental protocol was applied, support the notion that both receptors converge on the same population of K(ACh) channels. The observation illustrated in Fig. 3 might suggest that Gβγ binding site(s) on the channels subunits become saturated at a concentration of Gβγ, which is small compared with the concentration of free Gβγ that can be produced by activation of a large number of receptors. A myocyte transfected with either of the two receptor subtypes should contain native β1-AR, native β2-AR, and the overexpressed subtype. It is conceivable, therefore, that activation of IK(ACh) proceeds by the native receptors plus the overexpressed subtype, simply due to the fact that the loss in target specificity is related to the increase in total number of β-AR. Alternatively, activation of IK(ACh) could result from the overexpressed β-AR only, without a contribution of the native receptors, which might be precluded from activating IK(ACh)due to functional compartmentalization with other signaling components (5Xiao R.-P. Cheng H. Zhou Y.-Y. Kuschel M. Lakatta E.G. Circ. Res. 1999; 85: 1092-1100Crossref PubMed Scopus (218) Google Scholar, 37Neubig R.R. FASEB J. 1994; 8: 939-946Crossref PubMed Scopus (318) Google Scholar). We addressed this question by means of the β1-AR specific antagonist CGP20712A (CGP). As shown in Fig.4A, CGP at 10 μmdid not affect the current induced by 10 μm Iso in a β2-AR-transfected myocyte, suggesting that native β1-AR do not contribute to the response or, at least, that their contribution is below the detection limit. On the other hand, CGP completely inhibited the Iso-induced current in β1-AR-overexpressing cells (Fig. 4B); identity of the inhibited current with IK(ACh) was confirmed by the difference current voltage relation (Fig. 4C). This, in turn, suggests that native β2-AR are not involved in activation of IK(ACh), since their contribution should be resistant to this antagonist. Complete insensitivity of Iso-induced IK(ACh) to 10 μm CGP was observed in 4/4 β2-AR-overexpressing myocytes, whereas a complete block of Iso-induced IK(ACh)was found in 4/4 myocytes overexpressing β1-AR. An analogous experiment using a selective β2-AR antagonist (ICI118,551) could not be performed, since this compound, at concentrations >0.1 μm inhibited IK(ACh) induced by ACh (Fig.4D), presumably by a direct blocking action on the K(ACh) channel. A recent paper (21Mullner C. Vorobiov D. Bera A.K. Uezono Y. Yakubovich D. Frohnwieser-Steinecker B. Dascal N. Schreibmayer W. J. Gen. Physiol. 2000; 115: 547-558Crossref PubMed Scopus (50) Google Scholar) showed some interference of β-adrenergic stimulation with native and expressed GIRK channels, which was interpreted in terms of a PKA-mediated phosphorylation. Moreover, Ref.38Medina I. Krapivinsky G. Arnold S. Kovoor P. Krapivinsky L. Clapham D.E. J. Biol. Chem. 2000; 275: 29709-29716Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar demonstrated that protein phosphorylation in the C terminus of GIRK1 is conditional for Gβγ activation of GIRK1/GIRK4 channel complexes. Information on the signaling mechanism controlling GIRK1 phosphorylation, including the type of protein kinase involved, was not provided in that publication. On this background, it cannot be excluded that, in myocytes overexpressing β-ARs, apart from a Gβγ-mediated activation, a PKA-mediated effect contributes to activation of IK(ACh). To bypass Gβγ signaling, we used forskolin, a direct (receptor-independent) activator of PKA. Fig.5A demonstrates that forskolin (10 μm), applied for 90 s, in a myocyte overexpressing β2-AR failed to cause any measurable activation of IK(ACh), whereas Iso induced a robust inward current of 80% of the maximum current. This result is qualitatively representative for 10/10 non-transfected and 9/9 β-AR-transfected cells. In none of these cells did forskolin induce a measurable K+ current or altered properties of IK(ACh)activated by ACh. It has been shown previously that atrial myocytes isolated from guinea pig hearts and cultured under otherwise identical conditions respond to stimulation of the cAMP-dependent signaling pathway by Iso or forskolin, respectively, by a substantial increase in l-type Ca2+ current (ICa), a paradigmatic target for PKA in cardiac myocytes in general (Ref. 39Boller M. Pott L. Pflügers Arch. 1989; 415: 276-288Crossref PubMed Scopus (19) Google Scholar; see Ref. 40Pelzer D. Pelzer S. McDonald T.F. Rev. Physiol. Biochem. Pharmacol. 1990; 114: 107-207Crossref PubMed Google Scholar for review) and a sensitization of Ca2+-release from the sarcoplasmic reticulum. In order to confirm that the cAMP-dependent signaling pathway is also working in cultured rat myocytes under the present experimental conditions, we tested the effect of forskolin on ICa. In Fig. 5B volta" @default.
• W1989821836 created "2016-06-24" @default.
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• W1989821836 creator A5049593878 @default.
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• W1989821836 date "2001-10-01" @default.
• W1989821836 modified "2023-09-30" @default.
• W1989821836 title "Overexpression of β1 and β2Adrenergic Receptors in Rat Atrial Myocytes" @default.
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