Coupling of M2 Muscarinic Receptors to Membrane Ion Channels via Phosphoinositide 3-Kinase γ and Atypical Protein Kinase C

Abstract

We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or α2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gβγ stimulation of phosphoinositide 3-kinase γ (PI-3Kγ), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCζ at concentrations as low as 1 nm. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of βγ binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling. We report a novel signaling pathway linking M2 muscarinic receptors to metabotropic ion channels. Stimulation of heterologously expressed M2 receptors, but not other Gi/Go-associated receptors (M4 or α2c), activates a calcium- and voltage-independent chloride current in Xenopus oocytes. We show that the stimulatory pathway linking M2 receptors to these chloride channels consists of Gβγ stimulation of phosphoinositide 3-kinase γ (PI-3Kγ), formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activation of atypical protein kinase C (PKC). The chloride current is activated in the absence of M2 receptor stimulation by the injection of PIP3, and PIP3 current activation is blocked by a pseudosubstrate inhibitory peptide of atypical PKC but not other PKCs. Moreover, the current is activated by injection of recombinant PKCζ at concentrations as low as 1 nm. M2 receptor-current coupling was disrupted by inhibiton of PI-3K and by injection of βγ binding peptides, but it was not affected by expression of dominant negative p85 cRNA. We also show that this pathway mediates M2 receptor coupling to metabotropic nonselective cation channels in mammalian smooth muscle cells, thus demonstrating the broad relevance of this signaling cascade in neurotransmitter signaling. M2 muscarinic receptors mediate numerous cellular functions including presynaptic regulation of transmitter release by neurons in the brain and autonomic nervous system and postsynaptic control of the heart, smooth muscle, and secretory cells. Stimulation of M2 receptors in heart cells opens inward rectifier K+ channels through a direct interaction between released G protein βγ subunits and channel proteins (1Logothetis D.E. Kurachi Y. Galper J. Neer E.J. Clapham D.E. Nature. 1987; 325: 321-326Crossref PubMed Scopus (868) Google Scholar, 2Wickman K.D. Iniguez-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 (380) Google Scholar, 3Reuveny E. Slesinger P.A. Inglese J. Morales J.M. Iniguez-Lluhi J.A. Lefkowitz R.J. Bourne H.R. Jan Y.N. Jan L.Y. Nature. 1994; 370: 143-146Crossref PubMed Scopus (417) Google Scholar, 4Huang C.L. Slesinger P.A. Casey P.J. Jan Y.N. Jan L.Y. Neuron. 1995; 15: 1133-1143Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 5Slesinger P.A. Reuveny E. Jan Y.N. Jan L.Y. Neuron. 1995; 15: 1145-1156Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 6Kunkel M.T. Peralta E.G. Cell. 1995; 83: 443-449Abstract Full Text PDF PubMed Scopus (134) Google Scholar), but the signaling pathways linking M2 receptors to ion channels in nerve, smooth muscle, and secretory cells are poorly understood. Hormone-stimulated phosphoinositide 3-kinase (PI-3K) 1The abbreviations used are: PI-3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKC, protein kinase C; aPKC, atypical PKC; cPKC, conventional PKC; mACH, methacholine; GF109203X, bisindolylmaleimide I 1The abbreviations used are: PI-3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PKC, protein kinase C; aPKC, atypical PKC; cPKC, conventional PKC; mACH, methacholine; GF109203X, bisindolylmaleimide I plays an important role in cell growth, adhesion, and survival and in actin assembly (7Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1221) Google Scholar). The identification of the Gβγ-stimulated PI-3Kγ (8Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 9Thomason P.A. James S.K. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar, 10Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Science. 1995; 269: 690-693Crossref PubMed Scopus (639) Google Scholar) extends the potential range of processes mediated by PI-3K to G protein-coupled receptors, although specific physiological processes mediated by PI-3Kγ have not been identified. One potential target of lipid second messengers generated by PI-3K are atypical protein kinase C enzymes (aPKCs), which lack a diacylglycerol binding site and are activatedin vitro by phosphatidylinositol phosphates (11Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar, 12Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 13Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 14Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2356) Google Scholar). Here we show that stimulation of heterologously expressed M2receptors, but not other Gi/Go-linked receptors, opens endogenous metabotropic chloride channels inXenopus oocytes by activation of PI-3Kγ, generation of PIP3, and stimulation of aPKC. We also show that this signaling pathway mediates physiological coupling between M2 receptors and nonselective cation channels in mammalian smooth muscle cells. Surgical removal of oocytes was performed in the laboratory of Dr. Peter Drain in accordance with a protocol approved by the University of Pennsylvania Animal Care and Use Committee. Oocyte defolliculation, injection, and dual-electrode voltage clamp were as described previously (15Nara M. Dhulipala P.D. Wang Y.X. Kotlikoff M.I. J. Biol. Chem. 1998; 273: 14920-14924Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Currents were amplified (OC-725C, Warner Instruments, Hamden, CT), filtered at 200 Hz (−3 dB; 8-pole Bessel filter, model 902, Frequency Devices, Haverhill, MA), digitized at 1 kHz (TL125, Axon Instruments, Foster City, CA), and monitored and simultaneously stored on disk (Axotape, Axon Instruments). All currents shown were leak-subtracted using identical voltage paradigms before exposure to mACH. Pipettes with resistances between 0.5 and 1 megaohm were filled with 3 m KCl. Extracellular bath solution was (concentrations in mm): 115 NaCl, 2.8 KCl, 1 MgCl2, and 10 Hepes. Intracellular injection of all substances consisted of 50-nanoliter volumes, and the indicated concentrations assume a 20-fold dilution in the oocyte cytosol (1 μl volume). Injections were made 10 min before oocyte stimulation. Antibodies directed against specific Gα subunits were injected at the obtained concentration (1:20 final titer). Solution changes were made by washing the bath with at least 25× bath volume (1 ml). cRNA was prepared using the mMessage mMachine kit (Ambion). Plasmid DNAs were linearized with appropriate restriction enzymes, and cRNAs were synthesized using the appropriate RNA polymerase. The integrity of the cRNAs was tested on ethidium bromide-stained agarose gels, and concentrations were estimated by spectrophotometry. The ΔP85 construct (16Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar) in PGEX was obtained from Dr. M. Kasuga and subcloned in pBlueScript KS+ (Stratagene). M2, M3, and M4 clones were kindly provided by Dr. E. Peralta and Dr. T. Morelli. Equine trachealis tissue was obtained in accordance with protocols approved by the University of Pennsylvania Animal Care and Use Committee. Cell isolation, whole cell recording, and agonist application were as described previously (17Wang Y.-X. Kotlikoff M.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14918-14923Crossref PubMed Scopus (97) Google Scholar). Seals were formed with 3–5-megaohm pipettes, and cells were dialyzed with the following (concentrations in mm): 130 CsCl, 1.2 MgCl2, 1 MgATP, 0.1 EGTA, and 10 Hepes, pH 7.3. The bath solution was (concentrations in mm): 125 NaCl, 5 KCl, 1 MgSO4, 1.8 CaCl2, 10 glucose, and 10 Hepes, pH 7.4. Cells were allowed to adhere to a glass coverslip, and recordings in relaxed cells were made at room temperature. Following break-in, cells were dialyzed for 5 min before activation of currents by application of the muscarinic agonist using a puffer pipette. Chelerythrine, GF109203X, Gö 6976, and cPKC pseudosubstrate peptide were obtained from Calbiochem and PIP3 and PKCζ from Biomol. aPKC and QEHA peptides were custom synthesized. Bovine transducin α was kindly provided by Dr. H. E. Hamm. Heterologous expression of M2 receptors inXenopus oocytes indicated that receptor stimulation activates a novel metabotropic chloride current. The muscarinic agonist methacholine (mACH) activated only a sustained inward current in oocytes injected with M2 receptor cRNA and recorded in calcium-free conditions, whereas a transient inward current was observed in oocytes expressing Gq/11-coupled M3receptors (Fig. 1 A). The transient current was shown to be the ubiquitous endogenous calcium-activated chloride current, as it was blocked by chelating intracellular calcium or by inhibiting calcium release with heparin, whereas activation of the M2 current could be obtained repeatedly in calcium-free solutions and was not affected by intracellular calcium chelation or by blockade of calcium release. These currents were identified in early original experiments characterizing muscarinic acetylcholine receptor subtypes inXenopus oocytes (18Fukuda K. Kubo T. Akiba I. Maeda A. Mishina M. Numa S. Nature. 1987; 327: 623-625Crossref PubMed Scopus (117) Google Scholar), although the sustained current was not isolated and was reported to be cation selective. Ion substitution experiments clearly identified the M2 current as anion selective, with a selectivity sequence of I− > Cl− > isethionate, whereas substitution of more than 90% of the sodium for Tris had no effect on current reversal potential (Fig. 1, B and C). The chloride current activated following M2 receptor binding was voltage-independent, and no measurable current was available in the absence of M2stimulation (no shift in background current observed with anion substitution), indicating that receptor binding is required for channel opening. To examine the linkage between M2 receptors and the novel chloride current, we used antibodies, specific peptides, dominant negative constructs, and enzyme inhibitors to disrupt receptor-effector coupling. M2 receptor-chloride current coupling was blocked by preinjection of antibodies directed against Gαi or Gαo, but not Gαq, proteins (Fig.2, A and B). Anti-Gαi1/Gαi2 and anti-Gαi3/Gαo antibodies blocked 83 ± 3% (n = 6) and 52 ± 4% (n = 6) of the current, respectively. Whereas M2 signaling was clearly coupled by Gi/Go proteins, the M2 current was not activated by heterologously expressed adrenergic α2C receptors or muscarinic M4receptors, which coupled weakly to intracellular calcium release and the associated calcium-activated chloride current (Fig. 2 C). These receptors also preferentially associate with Gi/Go proteins (19Coupry I. Duzic E. Lanier S.M. J. Biol. Chem. 1992; 267: 9852-9857Abstract Full Text PDF PubMed Google Scholar, 20Felder C.C. FASEB J. 1995; 9: 619-625Crossref PubMed Scopus (452) Google Scholar), indicating that the signaling pathway leading to activation of the M2 chloride current discriminates between receptors signaling through pertussis toxin-sensitive G proteins. Moreover, whereas both M2 and M4 receptors are capable of activating inward rectifying K+ channels through βγ proteins (21Gadbut A.P. Riccardi D. Wu L. Hebert S.C. Galper J.B. J. Biol. Chem. 1996; 271: 6398-6402Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), the stimulatory pathway linking M2 receptors and chloride channels effectively distinguishes between these closely related receptors. Protein kinase C (PKC) molecules that are activated by diacylglycerol and calcium following stimulation of phospholipase C by G protein-coupled receptors have been implicated in M2receptor-ion channel coupling (22Mochida S. Kobayashi H. Neurosci. Lett. 1988; 86: 201-206Crossref PubMed Scopus (10) Google Scholar, 23Bernheim L. Beech D.J. Hille B. Neuron. 1991; 6: 859-867Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 24Marsh S.J. Trouslard J. Leaney J.L. Brown D.A. Neuron. 1995; 15: 729-737Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 25Cantrell A.R. Ma J.Y. Scheuer T. Catterall W.A. Neuron. 1996; 16: 1019-1026Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). M2 coupling to the novel chloride current was disrupted by exposure of oocytes to the nonselective protein kinase C inhibitor chelerythrine. However, GF109203X and Gö 6976 (not shown), more selective inhibitors of several conventional and novel PKC isoforms, had no effect on current activation (Fig. 3, A andB). None of the protein kinase C inhibitors affected M3 coupling to phospholipase C, and conversely, M2 receptor coupling was not affected by phospholipase C inhibition (Fig. 3 A). aPKCs that are activated by phosphatidylinositol 4,5-diphosphate, PIP3, andcis-fatty acids have been implicated as effectors in mitogen, apoptotic, and contractile signaling (11Ono Y. Fujii T. Ogita K. Kikkawa U. Igarashi K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3099-3103Crossref PubMed Scopus (413) Google Scholar, 12Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 13Liscovitch M. Cantley L.C. Cell. 1994; 77: 329-334Abstract Full Text PDF PubMed Scopus (311) Google Scholar, 26Lozano J. Berra E. Municio M.M. Diaz-Meco M.T. Dominguez Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar, 27Gailly P. Gong M.C. Somlyo A.V. Somlyo A.P. J. Physiol. (London). 1997; 500: 95-109Crossref Scopus (102) Google Scholar), although involvement of aPKCs in ion channel signaling has not been reported. We examined the role of protein kinase C subtypes in M2 receptor coupling using peptides that selectively bind and inhibit conventional PKCs or aPKCs. Preinjection of aPKC pseudosubstrate inhibitory peptides (28Dominguez I. Diaz-Meco M.T. Municio M.M. Berra E. Garcia de Herreros A. Cornet M.E. Sanz L. Moscat J. Mol. Cell. Biol. 1992; 12: 3776-3783Crossref PubMed Scopus (141) Google Scholar), but not cPKC inhibitory peptides (29House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (787) Google Scholar), inhibited the M2 chloride current coupling, suggesting that aPKC activation is necessary for M2-chloride channel coupling. We confirmed the role of aPKC in receptor-effector coupling by direct injection of aPKC into the cytosol (Fig.4). Injection of recombinant PKCζ activated the chloride current in a concentration dependent fashion, with currents observed at final concentrations of PKCζ as low as 1 nm. Moreover, injection of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which stimulates PKCζin vitro (12Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar), activated the current. Current activation was specific to PIP3 injection. Phosphatidylinositol 4,5-diphosphate, phosphatidic acid, phosphatidylcholine, linolenic acid, phosphatidylserine, and arachidonic acid failed to induce any current (data not shown). PIP3 and PKCζ currents were indistinguishable from that observed following M2 receptor stimulation in several respects. First, PIP3 and PKCζ activated an anion selective current with a permeability sequence of I− > Cl− > isethionate and a current-voltage relationship identical to the M2 current. Second, the current was sustained in the absence of extracellular calcium following activation by either agent and was insensitive to calcium chelation. Third, currents activated by M2 receptor stimulation and by PIP3 or PKCζ injection were not additive. Following activation of the current by 50 μmmACH, little or no further current was elicited by the injection of PIP3 or PKCζ (n = 6), and PIP3 or PKCζ, current activation abrogated subsequent mACH current (n = 5) (Fig. 4 A). Finally, the aPKC pseudosubstrate inhibitor blocked both the M2 and PIP3-induced chloride currents (Fig. 4, A andB). PIP3 has been shown to stimulate novel PKC-ε and A. M. R. S. A. D.J. Cantley L.C. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) as as atypical PKCζ and the of PIP3 currents by the aPKC pseudosubstrate peptide the specific of PKCs current Moreover, diacylglycerol activated the current weakly activated the calcium-activated chloride and current coupling was not affected by exposure to these (Fig. 4 A). and peptide inhibitors that are selective for aPKCs blocked activation of the M2 chloride current, and lipid of aPKCs not of conventional and novel and PKCζ the current in the absence of M2 receptor indicating that stimulation of aPKC is both necessary and to chloride channels. Hormone-stimulated PI-3K in the formation of PIP3, which has been implicated as a second in a of cellular processes as actin and A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1221) Google Scholar). of chloride channels by PIP3 the involvement of PI-3K in the stimulatory pathway linking M2 receptor stimulation to chloride channel opening. with a signaling the PI-3K inhibitor blocked the current with no effect on (not shown). of have been identified on activation by kinase or by G protein-coupled signaling The are of a that is following interaction with a p85 or and whereas G kinase through the direct activation of by the binding of Gβγ subunits P.A. James S.K. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar, 10Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnberg B. Science. 1995; 269: 690-693Crossref PubMed Scopus (639) Google Scholar, A. H. M. Cooke J. Smrcka M. K. P. Hawkins P.T. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Downes C.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, D. Hanck T. T. U. R. Nurnberg B. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). To the of the PI-3K in M2 coupling to aPKC, we used a peptide of 2 a Gβγ binding which Gβγ signaling to several protein J. M. J. A. J. J. D.J. J.L. Exton J.H. Inglese J. Lefkowitz R.J. D.E. R. Science. 1995; 268: PubMed Scopus Google Scholar) and has been shown to inhibit βγ stimulation of PI-3Kγ in vitro D. Hanck T. T. U. R. Nurnberg B. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The QEHA peptide inhibited coupling between M2 receptors and chloride channels by as as inhibition of coupling by the peptide was to that observed for PI-3Kγ D. Hanck T. T. U. R. Nurnberg B. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. A and B). The peptide had no effect on PIP3 activation of the current, indicating that the is of PIP3 formation (data not shown). transducin α proteins, which bind Gβγ subunits with inhibited M2-chloride channel coupling (Fig. 5 C). Injection of proteins to a final concentration of 5 inhibited the current by ± (n = whereas injection of transducin α proteins was We also expressed a dominant negative p85 cRNA which has been shown to the activation of PI-3K (16Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar). of or p85 had no effect on M2 signaling cRNAs were injected at concentrations to than M2 receptor cRNA. Finally, we to the coupling pathway was in physiological M2 of acetylcholine from stimulates M2 receptors on smooth muscle cells at in the and Stimulation of M2 receptors on isolated activates a sustained cation current that mediates postsynaptic R.J. Nature. PubMed Scopus Google Scholar, W.A. J. 1987; PubMed Scopus Google Scholar, Y.X. Kotlikoff M.I. J. Physiol. 1997; 273: PubMed Google Scholar, W.A. Wang J. Physiol. 1996; 271: PubMed Google Scholar), although the coupling is poorly understood. smooth muscle cells, of the aPKC pseudosubstrate peptide selectively I the calcium-activated chloride current that is associated with calcium release Y.X. Kotlikoff M.I. J. Physiol. 1997; 273: PubMed Google Scholar) (Fig. 6), indicating that aPKC activation is necessary for M2 coupling in smooth of the cPKC peptide had no effect on coupling. M2 coupling but not M3 coupling was also disrupted by of the QEHA peptide and by the kinase inhibitor indicating that of the signaling pathway linking to chloride channels in Xenopus oocytes are required for coupling of these receptors in mammalian cells. be that coupling of muscarinic receptors to nonselective cation is an of signaling in which generated by the stimulation of the M2 and M3 receptors are required for current activation Y.X. Kotlikoff M.I. J. Physiol. 1997; 273: PubMed Google Scholar, R. J. Physiol. (London). Scopus Google Scholar, P. J. Physiol. (London). 1991; Scopus Google Scholar). current activation the release of intracellular although release is not for channel M2 receptor Y.X. Kotlikoff M.I. J. Physiol. 1997; 273: PubMed Google Scholar). of with PIP3 not in the activation of I (data not shown). these that the coupling pathway physiological postsynaptic muscarinic signaling in smooth we a novel signaling pathway linking M2 receptors to metabotropic ion channels. binding in the stimulation of the PI-3Kγ, formation of PIP3, and activation of aPKC. signaling pathway to the of a second chloride current in Xenopus oocytes and mediates activation of nonselective cation channels in smooth muscle cells. These a novel signaling cascade linking G protein-coupled receptors to ion channels and further into the role of lipid second messengers in receptor signaling. We H. E. T. E. and M. Kasuga for