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• W2003971289 abstract "Gi protein-coupled receptors such as the M2 muscarinic acetylcholine receptor (mAChR) and A1 adenosine receptor have been shown to activate G protein-activated inwardlyrectifying K+ channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M2 receptors not only activate but also reversibly inhibit these K+currents when stimulated with agonist for up to 2 min. The M2 mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5′-O-(thiotriphosphate) in the pipette. Under these conditions the M2 mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gβγ, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gβγ subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M2 mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M2 mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M4 mAChR and α1A adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A1 and A3 adenosine receptors and α2A and α2C adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M2 and M4 mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gβγ-activated GIRK currents in a membrane-delimited fashion. Gi protein-coupled receptors such as the M2 muscarinic acetylcholine receptor (mAChR) and A1 adenosine receptor have been shown to activate G protein-activated inwardlyrectifying K+ channels (GIRKs) via pertussis toxin-sensitive G proteins in atrial myocytes and in many neuronal cells. Here we show that muscarinic M2 receptors not only activate but also reversibly inhibit these K+currents when stimulated with agonist for up to 2 min. The M2 mAChR-mediated inhibition of the channel was also observed when the channels were first activated by inclusion of guanosine 5′-O-(thiotriphosphate) in the pipette. Under these conditions the M2 mAChR-induced inhibition was quasi-irreversible, suggesting a role for G proteins in the inhibitory process. In contrast, when GIRK currents were maximally activated by co-expressing exogenous Gβγ, the extent of acetylcholine (ACh)-induced inhibition was significantly reduced, suggesting competition between the receptor-mediated inhibition and the large pool of available Gβγ subunits. The signaling pathway that led to the ACh-induced inhibition of GIRK channels was unaffected by pertussis toxin pretreatment. Furthermore, the internalization and agonist-induced phosphorylation of M2 mAChR was not required because a phosphorylation- and internalization-deficient mutant of the M2 mAChR was as potent as the wild-type counterpart. Pharmacological agents modulating various protein kinases or phosphatidylinositol 3-kinase did not affect the inhibition of GIRK currents. Furthermore, the signaling pathway that mediates GIRK current inhibition was found to be membrane-delimited because bath application of ACh did not inhibit GIRK channel activity in cell-attached patches. Other G protein-coupled receptors including M4 mAChR and α1A adrenergic receptors also caused the inhibition, whereas other G protein-coupled receptors including A1 and A3 adenosine receptors and α2A and α2C adrenergic receptors could not induce the inhibition. The presented results suggest the existence of a novel signaling pathway that can be activated selectively by M2 and M4 mAChR but not by adenosine receptors and that involves non-pertussis toxin-sensitive G proteins leading to an inhibition of Gβγ-activated GIRK currents in a membrane-delimited fashion. G protein-coupled receptor muscarinic acetylcholine receptor G protein-activated inwardly rectifying K+ channel pertussis toxin Chinese hamster ovary acetylcholine phenylephrine adrenergic receptor guanosine 5′-O-(thiotriphosphate) γ-aminobutyric acid, type B 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid atrial muscarinic K+ current human embryonic kidney An enormous variety of G protein-coupled receptors (GPCRs)1 allow for a large number of extracellular signals to converge on a relatively small number of heterotrimeric G proteins (1.Strader C.D. Fong T.M. Graziano M.P. Tota M.R. FASEB J. 1995; 9: 745-754Crossref PubMed Scopus (327) Google Scholar), which in turn cause activation of downstream effectors. When a cell receives multiple signals that are transduced through different GPCRs, the integration of multiple signaling events is complex not only because of the fact that the G proteins have multiple effector systems but also because of “cross-talk” between signaling pathways. Recently, evidence has emerged that cross-talk between GPCRs not only can occur between pathways that utilize different classes of G proteins but also within one G protein family (2.Polo-Parada L. Pilar G. J. Neurosci. 1999; 19: 5213-5227Crossref PubMed Google Scholar). The current study presents a new case of cross-talk between signals that are produced by activation of adenosine and muscarinic receptors that are known to couple to the same class of G proteins. The initial goal of this study was to investigate the regulation and the desensitization of M2 mAChR- and A1adenosine receptor-activated K+ channels that give rise to the current known as IKACh. IKACh channels were first identified in the supraventricular tissue of the heart, and their regulation by M2 mAChRs and other GPCRs, such as the A1 adenosine or lysosphingolipid receptors, has been the topic of many different studies over the past two decades (3.Sakmann B. Noma A. Trautwein W. Nature. 1983; 303: 250-253Crossref PubMed Scopus (314) Google Scholar, 4.Belardinelli L. Isenberg G. Am. J. Physiol. 1983; 244: H734-H737PubMed Google Scholar, 5.Bünemann M. Liliom K. Brandts B.K. Pott L. Tseng J.L. Desiderio D.M. Sun G. Miller D. Tigyi G. EMBO J. 1996; 15: 5527-5534Crossref PubMed Scopus (119) Google Scholar). IKACh channels were characterized as Gprotein-activated inwardly rectifyingK+ channels (GIRKs) that are activated by pertussis toxin (PTX)-sensitive G proteins in a membrane-delimited fashion (3.Sakmann B. Noma A. Trautwein W. Nature. 1983; 303: 250-253Crossref PubMed Scopus (314) Google Scholar, 6.Soejima M. Noma A. Pfluegers Arch. Eur. J. Physiol. 1984; 400: 424-431Crossref PubMed Scopus (293) Google Scholar, 7.Breitwieser G.E. Szabo G. Nature. 1985; 317: 538-540Crossref PubMed Scopus (356) Google Scholar, 8.Dascal N. Cell. Signal. 1997; 9: 551-573Crossref PubMed Scopus (268) Google Scholar, 9.Wickman K. Clapham D.E. Physiol. Rev. 1995; 75: 865-885Crossref PubMed Scopus (345) Google Scholar). It has become clear that GIRK channels are activated upon binding of the Gβγ subunits to the channel (10.Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (866) Google Scholar, 11.Krapivinsky G. Krapivinsky L. Wickman K. Clapham D.E. J. Biol. Chem. 1995; 270: 29059-29062Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 12.Krapivinsky G. Kennedy M.E. Nemec J. Medina I. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 16946-16952Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). GIRK channels are heteromultimers of two homologous subunits. GIRK1 and GIRK4 form the cardiac channel (13.Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (753) Google Scholar, 14.Corey S. Krapivinsky G. Krapivinsky L. Clapham D.E. J. Biol. Chem. 1998; 273: 5271-5278Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), whereas GIRK1 and GIRK2 or GIRK3 form certain neuronal channels (15.Lesage F. Duprat F. Fink M. Guillemare E. Coppola T. Lazdunski M. Hugnot J.P. FEBS Lett. 1994; 353: 37-42Crossref PubMed Scopus (268) Google Scholar, 16.Kofuji P. Davidson N. Lester H.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6542-6546Crossref PubMed Scopus (269) Google Scholar). The GIRK channels have been functionally expressed in a variety of cells includingXenopus oocytes and mammalian HEK and CHO cells (13.Krapivinsky G. Gordon E.A. Wickman K. Velimirovic B. Krapivinsky L. Clapham D.E. Nature. 1995; 374: 135-141Crossref PubMed Scopus (753) Google Scholar,17.Kubo Y. Reuveny E. Slesinger P.A. Jan Y.N. Jan L.Y. Nature. 1993; 364: 802-806Crossref PubMed Scopus (544) Google Scholar, 18.Dascal N. Schreibmayer W. Lim N.F. Wang W. Chavkin C. DiMagno L. Labarca C. Kieffer B.L. Gaveriaux-Ruff C. Trollinger D. Lester H.A. Davidson N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10235-10239Crossref PubMed Scopus (337) Google Scholar, 19.Bünemann M. Hosey M.M. J. Biol. Chem. 1998; 273: 31186-31190Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Desensitization of GPCR-activated GIRK currents has been observed in native tissues and in heterologous expression systems. We have utilized the GIRK channels as a readout system to study desensitization of G protein-mediated signals for two reasons. First, electrophysiological recording of GIRK currents with the patch clamp technique allows for real time measurement of G protein-mediated signals in intact cells. Second, the GIRK channels conduct potassium ions, which diffuse very rapidly, and the concentration of K+ can be controlled; therefore, even after activation of GIRK channels for a long period of time, depletion of intra- or extracellular K+ does not occur, and hence desensitization at the level of the channels does not play a major role in desensitization events. Desensitization of GPCR-generated signals can be homologous, where only the stimulated GPCR desensitizes, or heterologous, where the signaling by heterologous GPCRs is inhibited. To distinguish between homologous and heterologous desensitization processes, two different Gi-coupled receptors (A1 adenosine and M2 mAChR) were co-transfected into HEK293 cells with GIRK1 and GIRK4. Before and after an initial treatment of one of these receptors with agonist, the response to stimulation of the other receptor was determined. If the response of the treated receptor only was reduced after the initial treatment with agonist, this was considered homologous desensitization. However, if there was a reduction of the response of the untreated receptor after an initial treatment of the other receptor, this was considered to reflect heterologous desensitization. We report novel observations concerning heterologous desensitization of GPCR-activated GIRK channels. Chinese hamster ovary (CHO-K1) cells (a gift from Dr. J. Lomasney) were grown in Ham's F-12 medium (Life Technologies, Inc.). The medium was supplemented with 10% fetal bovine serum and streptomycin/penicillin (100 units each). Cells were grown under 7% CO2 at 37 °C. In all transfections for electrophysiological studies, the CD8 reporter gene system was used to visualize transfected cells (20.Jurman M.E. Boland L.M. Liu Y. Yellen G. BioTechniques. 1994; 17: 876-881PubMed Google Scholar). Dynabeads coated with anti-CD8 antibodies were purchased from Dynal. The cells were transfected as described below with various plasmids from the following list: human M2 mAChR (in pcDNA3, 0.8 μg), M2Δ1 (in pCR3, 0.8 μg) (21.Pals-Rylaarsdam R. Xu Y. Witt-Enderby P. Benovic J.L. Hosey M.M. J. Biol. Chem. 1995; 270: 29004-29011Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), M4 mAChR and M3 mAChR (both in pcDNA3, 0.8 μg each) (gifts from Dr. E. Peralta), human A1 or A3 adenosine receptors (in CLDN 10B, 0.2 μg; gifts from Dr. J. Linden), human α2A or α2C adrenergic receptors (in pcDNA3.1, 0.5 μg; gifts from Dr. L. Hein), human α1A adrenergic receptors (in pcCMV5, 0.5 μg; a gift from Dr. J. Lomasney), mouse GIRK1 (in pC1, 0.3 μg; a gift from Dr. F. Lesage), mouse GIRK4 (in pcDNA1, 0.3 μg; a gift from Dr. F. Lesage), human Gβ1 (in pCMV5, 0.3 μg; a gift from Dr. H. A. Bourne), human Gγ2 (in pcDNA1, 0.3 μg; a gift from Dr. H. A. Bourne), and human CD8 (in H3, 0.15 μg; a gift from Dr. G. Yellen). The transfection method used in this study was based on a method (22.Forsayeth J.R. Garcia P.D. BioTechniques. 1994; 17: 354-358PubMed Google Scholar) in which replication-deficient adenoviruses lacking the E1 gene were coated with DEAE-dextran and mammalian expression vectors to import the cDNAs of interest. Ad5dl312 viruses (a gift from Dr. K. Rundell) were allowed to replicate in HEK293 cells and were harvested when 70–90% of the HEK293 cells were rounded up. The cells were centrifuged gently, and the pellet was resuspended in 1 ml of Ham's F-12 medium/10-cm dish of cells. For cell lysis, cells were frozen and thawed three times and centrifuged. The supernatant was used as the viral stock solution. The viral stock (10 μl) was diluted with 130 μl of Ham's F-12 medium, and 80 μg of DEAE-dextran was added. The mixture was incubated at room temperature for 5–10 min. The total amounts of plasmid DNA (2.2 μg) were added (empty pcDNA 3.1 was used to balance cDNA amounts), and the mixture was incubated for another 2–5 min. Ham's F-12 medium (2 ml) was added, and this transfection solution was added onto one plate of 60–70% confluent CHO K1 cells. Prior to the incubation the cells were washed three times with 3 ml of serum and antibiotic-free medium. All assays were performed 48–72 h posttransfection. For the measurement of K+ currents an extracellular solution of the following composition was used (mm, pH 7.4): NaCl, 120; KCl, 20; CaCl2, 2; MgCl2, 1; Hepes-NaOH, 10. The internal (pipette) solution contained (mm): potassium glutamate, 100; KCl, 40; MgATP, 5; Hepes-KOH, 10; NaCl, 5; EGTA, 2; MgCl2, 1; GTP, 0.01 (pH 7.4). All standard salts as well as acetylcholine (ACh), adenosine (Ado), and phenylephrine (PhE) were from Sigma. Membrane currents were recorded under voltage clamp using conventional whole cell-patch clamp techniques (23.Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15098) Google Scholar). Patch pipettes were fabricated from borosilicate glass capillaries (GF-150–10, Warner Instruments) using a horizontal puller (P-95, Fleming & Poulsen). The DC resistance of the filled pipettes ranged from 2 to 5 megohms. Membrane currents were recorded using a patch clamp amplifier (Axopatch 200, Axon Instruments). Signals were analog-filtered using a low-pass Bessel filter (1–3 kHz corner frequency). Data were digitally stored using an IBM-compatible PC equipped with a hardware/software package (ISO2 by MFK, Frankfurt/Main, Germany) for voltage control, data acquisition, and data evaluation. IKACh was measured as an inward current using a holding potential of −90 mV as described (24.Bünemann M. Brandts B. zu Heringdorf D.M. van Koppen C.J. Jakobs K.H. Pott L. J. Physiol. (Lond.). 1995; 489: 701-777Crossref Scopus (109) Google Scholar). Voltage ramps (from −120 mV to +60 mV in 500 ms, every 10 s) were used to determine current-voltage relationships. M2 mAChRs, as well as A1adenosine receptors, can activate inwardly rectifying potassium currents carried by GIRK channels when the receptors and channel subunits are transiently co-expressed in CHO K1 cells. Agonist-activated currents were measured, using the whole cell-patch clamp configuration, as inward currents by setting the membrane potential negative (−90 mV) to the potassium equilibrium potential of about −50 mV (20 mm external potassium). In cells transfected with the GIRK subunits and the M2 mAChRs, application of ACh (1 μm) caused an activation of GIRK currents that rapidly desensitized during a 2-min application of ACh (Fig. 1 A, left panel). In contrast, in cells transfected with the GIRK subunits and the A1 adenosine receptors, rapid superfusion of the cells with adenosine (1 μm) induced an inwardly rectifying K+ current that exhibited only a marginal desensitization after a 2-min application of adenosine, and after washout of the agonist, GIRK currents deactivated to basal levels (Fig.1 A, right panel). To determine whether the desensitization of the ACh response took place at the level of the M2 mAChR or downstream of it, we analyzed the effects of a series of successive exposures to Ado and ACh in cells co-expressing both the A1 adenosine receptors and the M2 mAChRs (Fig. 1 B). An initial exposure to adenosine induced GIRK currents that were stable after an initial rapid, small desensitization (this type of desensitization is not the topic of this study but was studied in recent papers (25.Chuang H.H., Yu, M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11727-11732Crossref PubMed Scopus (107) Google Scholar, 26.Vorobiov D. Levin G. Lotan I. Dascal N. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 56-68Crossref PubMed Scopus (25) Google Scholar, 27.Shui Z. Boyett M.R. Zang W.J. J. Physiol. (Lond.). 1997; 505: 77-93Crossref Scopus (27) Google Scholar)). After washout of Ado and a return of currents to basal levels, the cells were exposed to ACh. The ACh-induced currents were of similar amplitude compared with those induced by Ado; however, again the ACh-induced GIRK currents markedly desensitized during the 2-min exposure to ACh. To test if the ACh treatment would affect subsequent effects of Ado, we exposed the cells to a second application of Ado within 1 min after the washout of ACh (Fig. 1 B). These Ado-elicited GIRK currents were substantially reduced compared with the initial Ado response (Fig.1 B), demonstrating that the prior treatment with ACh caused an inhibition of the subsequent Ado response. That the ACh-induced reduction of the Ado response was attributable to the preceding stimulation of M2 mAChR receptors was demonstrated by the observation that in cells lacking the M2 mAChRs, no inhibition of the Ado response by pretreatment with ACh was observed (Fig. 1 C). In addition, prior treatment with Ado did not substantially reduce the effects of a second stimulus with Ado (Fig.1 C), indicating that the activation of GIRK currents itself was not sufficient to cause desensitization. Furthermore, that prior application of Ado was not responsible for the M2mAChR-induced desensitization and inhibition of the GIRK currents was demonstrated by results showing similar responses elicited by the M2 mAChR in cells not previously stimulated with Ado (Fig.1 A and data not shown). The summarized results from a series of similar experiments demonstrated that prior exposure to ACh caused a reduction of the subsequent Ado response to 56 ± 4% (n = 13) of the initial Ado response, whereas prior exposure to Ado only caused a reduction of 5 ± 2.2% (n = 7) (Fig. 1 D). The results suggested that activation of the mAChRs caused an initial activation followed by an inhibition of GIRK currents and that the inhibition persisted during a subsequent exposure to Ado and thus diminished the response to Ado. To test if the ACh-induced inhibition of the Ado currents was reversible, we performed similar experiments as described above, in which we tested the effects of successive treatments with Ado at different times following a desensitizing treatment with ACh. The cells were co-transfected with the GIRKs and both receptors and initially exposed to a brief pulse of Ado, which activated the currents (Fig.2). After washout of Ado, the cells were exposed to ACh for 2 min during which there was an initial activation of the GIRK currents to ∼75% of the extent observed with Ado, followed by a substantial inhibition (Fig. 2). Immediately following washout of the ACh the cells were exposed briefly to Ado, and the response was substantially reduced compared with the initial Ado response (Fig. 2). However, the size of the Ado-induced currents increased over time following the washout of ACh, as the subsequent treatments with Ado resulted in larger currents. After 6 min following the washout of ACh, the response to Ado was almost comparable with that of the initial Ado response (Fig. 2, last trace). These results demonstrated that the ACh-induced inhibition of the currents was reversible, albeit with a relatively slow time scale (t 12 = 2–5 min). To test whether the ACh-induced inhibition of the currents was due to cross-desensitization of A1 receptors and whether GTP-binding proteins were participating in the inhibitory pathway, we performed experiments in which we activated the currents by preactivating the Gi proteins with GTPγS added to the pipette solution. This strategy allowed us to eliminate the need to first activate the currents with Ado, thus bypassing the Ado receptor, and allowed us to ask whether ACh could inhibit currents activated directly by G proteins. The amplitude of the GTPγS-activated GIRK currents was determined by a short application of 0.5 mmBa2+, which is sufficient to completely inhibit GIRK currents but does not significantly affect endogenous currents and therefore is used to define the base line (28.Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Crossref PubMed Scopus (658) Google Scholar) (Fig.3). The difference in currents before and after Ba2+ reflected the amplitude of the GTPγS-activated currents, and it can be seen in Fig. 3 that GTPγS caused a substantial activation of the currents. After a complete washout of Ba2+ and the return of the currents to the activated state, the cell was superfused with ACh for 1 min. Surprisingly, following an initial small, further activation of the currents, the activation of the M2 mAChRs by ACh caused a marked inhibition of the GTPγS-induced K+ currents (Fig. 3). The extent of inhibition of the GTPγS-activated currents by ACh was 57.5 ± 7.2% (n = 4, summarized in Fig.4 C). Moreover, in contrast to experiments performed without GTPγS (for example Fig. 2), no recovery from the ACh-induced inhibition of the GIRK currents was observed, even after 20 min after washout of ACh (Fig. 3). The inhibition of GTPγS-activated GIRK currents by ACh suggested that the ACh-induced inhibition was due to an inhibitory effect at the level of the G proteins or GIRKs. In addition, the results suggested that the inhibition of the Ado-induced GIRK currents by the M2mAChRs (Figs. 1 B and 2) was probably not due to a cross-desensitization of the A1 adenosine receptors. To avoid confusion we refer to this phenomenon as “ACh-induced inhibition of the GIRK currents” rather than heterologous desensitization. The observation that the M2 mAChR-induced inhibition of the GIRK currents became quasi-irreversible in the presence of intracellular GTPγS strongly suggested the involvement of GTP-binding proteins in this inhibitory pathway, as the poorly hydrolyzed GTPγS should lead to long lasting activation of GTP-dependent processes.Figure 4Inhibition of GIRK currents constitutively activated by heterologously expressed Gβ1γ2by activation of M2 mAChR. CHO cells were transfected with cDNAs encoding for Gβ1 and Gγ1subunits as well as GIRK1/4 and M2 mAChR. A, basal GIRK current levels under these conditions were determined by brief applications of Ba2+ (the arrow at theright of the current trace indicates current level in the presence of Ba2+). Addition of ACh did not result in any further current increase, indicating that GIRK channels were fully activated by co-expressed Gβγ. However, during the 2-min application of ACh a moderate inhibition of the GIRK currents was observed. B, characteristics of the ACh-inhibited current as determined by subtracting the current-voltage curves obtained prior to (A, a) and during (A, b) the application of ACh. C, summarized data of the inhibition (expressed as % inhibition) of GIRK currents either activated by GTPγS (as described in Fig. 3) or by heterologously expressed Gβγ in response to ACh for 2 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further define where the inhibition of GIRK currents by M2 mAChR occurred, we studied the M2 mAChR-induced inhibition of GIRK channels that were prestimulated with heterologously expressed Gβγ subunits. CHO cells that were transiently transfected with M2 mAChR, GIRK1, GIRK4, and Gβ1γ2 exhibited constitutively activated GIRK currents that were blocked by 0.5 mm Ba2+ (Fig. 4 A). The currents obtained with Gβγ expression were activated to a much larger degree than with receptor activation or GTPγS (compare Figs. 1, 2, and 3) and presumably reflected full activation of the channels. Consistent with this notion, stimulation of M2 mAChR with ACh did not cause any further increase in Ba2+-sensitive GIRK currents (Fig. 4 A). Notably, in most cases ACh still caused a reduction of basal currents (Fig. 4 A). These ACh-inhibited currents were found to have identical current-voltage relationships as GIRK currents (Fig. 4 B), indicating that the Gβγ-induced channel activity was inhibited by stimulation of M2 mAChR. However, the ACh-induced inhibition of GIRK currents in the presence of expressed Gβγ was significantly less (20 ± 3.8%,n = 8 after a 2-min application of 1 μmACh) compared with GIRK currents activated by GTPγS and endogenous G proteins (Fig. 4 C). Because these results were obtained under conditions in which full activation of the channels was induced by overexpression of Gβγ, the results suggested that the inhibitory effect of the M2 mAChRs on GIRK currents was due to a reduced interaction between the G proteins and the channels. The smaller inhibitory effect observed under these conditions presumably reflected competition between the receptor-induced inhibitory signal and the large pool of active Gβγ subunits. Because GTP-binding proteins seem to be involved in mediating the inhibitory effect of M2 mAChR on GIRK currents (Fig. 3), we tested whether PTX-sensitive G proteins were involved. To do this, transfected CHO cells expressing Gβγ subunits, GIRK1, GIRK4, and M2mAChR were pretreated with 200 ng/ml PTX for 4–6 h and subsequently tested for an ACh-induced inhibition of the Gβγ-activated GIRK currents (Fig. 5). The PTX pretreatment did not prevent or reduce the inhibitory effect of M2 mAChR on GIRK currents (inhibition of Ba2+-sensitive GIRK currents after a 2-min incubation with 1 μm ACh was 17.5 ± 6.6%, n = 4). In cells in which Gβγ subunits were not co-expressed, the PTX pretreatment completely blocked activation of GIRK currents by A1 receptors or M2 mAChR (n = 4, data not shown), indicating that the PTX treatment was 100% effective in blocking receptor-mediated activation of Gi and Goproteins. Thus, the inhibitory effects of the M2 mAChR on the GIRK channels, in contrast to their stimulatory effects, did not appear to involve a PTX-sensitive G protein. In addition, pretreatment with PTX did not alter the time course of recovery from inhibition after removal of ACh (data not shown). To test whether other GPCRs could also inhibit GIRK currents, M3 and M4 mAChRs were tested as well as A3 purinergic receptors and α2A and α2C adrenergic receptors. The M4 mAChRs, which, like the M2mAChRs are known to couple to PTX-sensitive G proteins, were as effective as M2 mAChRs in activating GIRK currents and in inducing the inhibition of GIRK currents upon treatment of transfected cells for 2 min with ACh (Fig. 6). Furthermore, activation of the M4 mAChRs caused an inhibition of a subsequent activation of the A1 receptors in a manner that was very similar to that caused by the M2mAChRs (compare Figs. 1 B and 6). In contrast, the other Gi/Go-linked GPCRs tested, namely the α2A and α2C adrenergic receptors and the A3 adenosine receptors, exhibited no obvious inhibitory effect on GIRK currents and basically behaved the same as A1 adenosine receptors (n = 5, data not shown). On the other hand, activation of M3 mAChRs, which activate Gq proteins rather than PTX-sensitive G proteins, caused a strong inhibition of Ado-activated GIRK currents. The inhibition of GIRK currents by the M3 mAChRs was not blocked by PTX in experiments similar to those described in Fig. 5. To test whether the inhibitory action on GIRK currents was specific for muscarinic receptors, the α1A adrenergic receptor (AR), another Gq-coupled receptor, was tested. Whereas activation of α1A adrenergic receptors with phenylephrine caused a very modest activation of the GIRK currents compared with Ado (Fig.7 A), these receptors caused a robust inhibition of GIRK currents as observed by the striking reduction in the ability of subsequent pulses of Ado to activate the currents (Fig. 7 A). Thus the inhibition of the GIRKs was observed with several subtypes of the mAChRs as well as with the α1A adrenergic receptor.Figure 7The inhibition of GIRK currents could be mediated by Gq-dependent and -independent pathways. CHO cells were transiently transfected with cDNAs for M2 mAChRs, A1 Ado receptors, GIRK1/4, and the Gq-coupled α1A ARs. A, experiments similar to those described in Figs. 1 B and 2were performed except that the AR agonist" @default.
• W2003971289 created "2016-06-24" @default.
• W2003971289 creator A5002203462 @default.
• W2003971289 creator A5030534164 @default.
• W2003971289 creator A5046805274 @default.
• W2003971289 creator A5067150916 @default.
• W2003971289 date "2000-04-01" @default.
• W2003971289 modified "2023-10-17" @default.
• W2003971289 title "Novel Inhibition of Gβγ-activated Potassium Currents Induced by M2 Muscarinic Receptors via a Pertussis Toxin-insensitive Pathway" @default.
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