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The expanding roles and mechanisms of G protein–mediated presynaptic inhibition

  • Zack Zurawski
    Footnotes
    Affiliations
    Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600

    Department of Anatomy and Cell Biology, University of Illinois, Chicago, Illinois 60612-7308
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  • Yun Young Yim
    Affiliations
    Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600
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  • Simon Alford
    Affiliations
    Department of Anatomy and Cell Biology, University of Illinois, Chicago, Illinois 60612-7308
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  • Heidi E. Hamm
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, 442 Robinson Research Bldg., 23rd Ave. South @ Pierce, Nashville, TN 37232-6600. Tel.: 615-343-3533; Fax: 615-343-1084
    Affiliations
    Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600
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  • Author Footnotes
    1 Present address: Dept. of Anatomy and Cell Biology, University of Illinois, Chicago, Illinois 60612-7308.
Open AccessPublished:February 01, 2019DOI:https://doi.org/10.1074/jbc.TM118.004163
      Throughout the past five decades, tremendous advancements have been made in our understanding of G protein signaling and presynaptic inhibition, many of which were published in the Journal of Biological Chemistry under the tenure of Herb Tabor as Editor-in-Chief. Here, we identify these critical advances, including the formulation of the ternary complex model of G protein–coupled receptor signaling and the discovery of Gβγ as a critical signaling component of the heterotrimeric G protein, along with the nature of presynaptic inhibition and its physiological role. We provide an overview for the discovery and physiological relevance of the two known Gβγ–mediated mechanisms for presynaptic inhibition: first, the action of Gβγ on voltage-gated calcium channels to inhibit calcium influx to the presynaptic active zone and, second, the direct binding of Gβγ to the SNARE complex to displace synaptotagmin downstream of calcium entry, which has been demonstrated to be important in neurons and secretory cells. These two mechanisms act in tandem with each other in a synergistic manner to provide more complete spatiotemporal control over neurotransmitter release.

      Introduction

      The development and characterization of selective agonists and antagonists for cell surface receptors was instrumental to formulating the current model of G protein receptor signaling. Radioligand antagonist-binding studies for numerous cell surface receptors, including the α- and β-adrenergic receptors (
      • Tsai B.S.
      • Lefkowitz R.J.
      Agonist-specific effects of guanine nucleotides on α-adrenergic receptors in human platelets.
      • Maguire M.E.
      • Van Arsdale P.M.
      • Gilman A.G.
      An agonist-specific effect of guanine nucleotides on binding to the β adrenergic receptor.
      ,
      • Lefkowitz R.J.
      • Mullikin D.
      • Caron M.G.
      Regulation of β-adrenergic receptors by guanyl-5′-yl imidodiphosphate and other purine nucleotides.
      ,
      • Stadel J.M.
      • DeLean A.
      • Lefkowitz R.J.
      A high affinity agonist-β-adrenergic receptor complex is an intermediate for catecholamine stimulation of adenylate cyclase in turkey and frog erythrocyte membranes.
      ,
      • Lucas M.
      • Bockaert J.
      Use of (−)-[3H]dihydroalprenolol to study β adrenergic receptor-adenylate cyclase coupling in C6 glioma cells: role of 5′-guanylylimidodiphosphate.
      ,
      • Limbird L.E.
      • Gill D.M.
      • Lefkowitz R.J.
      Agonist-promoted coupling of the β-adrenergic receptor with the guanine nucleotide regulatory protein of the adenylate cyclase system.
      • Hegstrand L.R.
      • Minneman K.P.
      • Molinoff P.B.
      Multiple effects of guanosine triphosphate on β adrenergic receptors and adenylate cyclase activity in rat heart, lung and brain.
      ), muscarinic acetylcholine receptors (
      • Berrie C.P.
      • Birdsall N.J.M.
      • Burgen A.S.V.
      • Hulme E.C.
      Guanine nucleotides modulate muscarinic receptor binding in the heart.
      ), dopaminergic receptors (
      • Zahniser N.R.
      • Molinoff P.B.
      Effect of guanine nucleotides on striatal dopamine receptors.
      ), and glucagon receptors (
      • Rodbell M.
      • Krans H.M.J.
      • Pohl S.L.
      • Birnbaumer L.
      The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver: IV. effects of guanyl nucleotides on binding of 125I-glucagon.
      ), demonstrated the existence of two affinity states within the receptor for agonist binding, a high-affinity state and a low-affinity state, but only a single state with respect to antagonist binding. This critical finding enabled Robert Lefkowitz to formulate and propose a ternary complex model in JBC using early computer-modeling techniques, where a high-affinity state for agonist binding consisting of the agonist, the receptor, and a yet-undetermined guanine nucleotide–binding component was needed for downstream adenylate cyclase activation (
      • De Lean A.
      • Stadel J.M.
      • Lefkowitz R.J.
      A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptor.
      ).
      The guanine nucleotide–binding component, known today as the heterotrimeric G protein, previously thought of as the “regulatory subunit” of adenylyl cyclase, was then purified in sufficiently large quantities to study. It was shown to consist of three polypeptides, one of which bound guanine nucleotides (
      • Northup J.K.
      • Sternweis P.C.
      • Smigel M.D.
      • Schleifer L.S.
      • Ross E.M.
      • Gilman A.G.
      Purification of the regulatory component of adenylate cyclase.
      ,
      • Sternweis P.C.
      • Northup J.K.
      • Smigel M.D.
      • Gilman A.G.
      The regulatory component of adenylate cyclase. Purification and properties.
      ). The isolation of purified β-adrenergic receptors (
      • Shorr R.G.
      • Heald S.L.
      • Jeffs P.W.
      • Lavin T.N.
      • Strohsacker M.W.
      • Lefkowitz R.J.
      • Caron M.G.
      The β-adrenergic receptor: rapid purification and covalent labeling by photoaffinity crosslinking.
      ,
      • Shorr R.G.
      • Strohsacker M.W.
      • Lavin T.N.
      • Lefkowitz R.J.
      • Caron M.G.
      The β1-adrenergic receptor of the turkey erythrocyte: molecular heterogeneity revealed by purification and photoaffinity labeling.
      • Shorr R.G.
      • Lefkowitz R.J.
      • Caron M.G.
      Purification of the β-adrenergic receptor: identification of the hormone binding subunit.
      ) and heterotrimeric G protein enabled the determination that the presence of these two components was necessary and sufficient for inducing two-state agonist binding in the receptor and nucleotide exchange for the G protein (
      • Brandt D.R.
      • Asano T.
      • Pedersen S.E.
      • Ross E.M.
      Reconstitution of catecholamine-stimulated guanosine triphosphatase activity.
      ,
      • Cerione R.A.
      • Codina J.
      • Benovic J.L.
      • Lefkowitz R.J.
      • Birnbaumer L.
      • Caron M.G.
      Mammalian β2-adrenergic receptor: reconstitution of functional interactions between pure receptor and pure stimulatory nucleotide binding protein of the adenylate cyclase system.
      ). Building upon this model, it was the purification of adenylate cyclase that enabled several articles in JBC and elsewhere from Lefkowitz, Gilman, and Smigel. These articles were instrumental for the initial model of GPCR
      The abbreviations used are: GPCR
      G protein–coupled receptor
      PAD
      primary afferent depolarization
      BNST
      bed nuclei of the stria terminals
      AR
      adrenergic receptor
      mGluR
      metabotropic glutamate receptor
      EPSC
      epithelial stem cell
      5-HT
      5-hydroxytryptamine.
      signaling, with critical reconstitution experiments showing that a minimum of three cellular proteinaceous complexes—the β adrenergic receptor, the heterotrimeric G protein, and the adenylate cyclase—were required for cAMP accumulation in response to agonists (
      • Cerione R.A.
      • Sibley D.R.
      • Codina J.
      • Benovic J.L.
      • Winslow J.
      • Neer E.J.
      • Birnbaumer L.
      • Caron M.G.
      • Lefkowitz R.J.
      Reconstitution of a hormone-sensitive adenylate cyclase system: the pure β-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit.
      ,
      • May D.C.
      • Ross E.M.
      • Gilman A.G.
      • Smigel M.D.
      Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins.
      • Ross E.M.
      • Gilman A.G.
      Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes.
      ). This effectively reproduced the cellular response in a strictly defined system.

      The discovery of Gβγ as a signaling molecule

      This initial model of GPCR signaling was predicated upon the GTP-bound Gα subunit conveying the transduced signal to the effector, whereas the Gβγ subunit’s only role was to obstruct the Gα effector-binding domains in the absence of ligand or act as an anchor to the plasma membrane (
      • Bourne H.R.
      GTP-binding proteins: One molecular machine can transduce diverse signals.
      ). An abrupt challenge to the model came from the critical finding of David Clapham’s group (
      • Logothetis D.E.
      • Kurachi Y.
      • Galper J.
      • Neer E.J.
      • Clapham D.E.
      The βγ subunits of GTP-binding proteins activate the muscarinic K+ channel in heart.
      ) that purified Gβγ subunits from the brain, and not Gα subunits, were responsible for transducing the signal of muscarinic acetylcholine receptors to activate GIRK channels in cardiac pacemaker and atrial cells. This novel finding was vehemently disputed by the groups of Birnbaumer and Brown (
      • Yatani A.
      • Codina J.
      • Imoto Y.
      • Reeves J.P.
      • Birnbaumer L.
      • Brown A.M.
      A G protein directly regulates mammalian cardiac calcium channels.
      ,
      • Codina J.
      • Yatani A.
      • Grenet D.
      • Brown A.M.
      • Birnbaumer L.
      The α subunit of the GTP binding protein Gk opens atrial potassium channels.
      • Kirsch G.E.
      • Yatani A.
      • Codina J.
      • Birnbaumer L.
      • Brown A.M.
      α-Subunit of Gk activates atrial K+ channels of chick, rat, and guinea pig.
      ), who showed evidence that the activating component of the heterotrimer, then termed “Gk” after the potassium channel, was a Gα subunit and that the purified Gβγ preparation of Clapham’s group may have been slightly contaminated with a high-affinity Gα, or interference was generated somehow from CHAPS detergent. The initial finding of Clapham’s group was echoed by other groups (
      • Kurachi Y.
      • Ito H.
      • Sugimoto T.
      • Katada T.
      • Ui M.
      Activation of atrial muscarinic K+ channels by low concentrations of βγ subunits of rat brain G protein.
      ). The dispute persisted until the work of Jan and Lester (
      • Reuveny 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.
      Activation of the cloned muscarinic potassium channel by G protein βγ subunits.
      ,
      • Lim N.F.
      • Dascal N.
      • Labarca C.
      • Davidson N.
      • Lester H.A.
      A G protein-gated K channel is activated via β2-adrenergic receptors and Gβγ subunits in Xenopus oocytes.
      • Kofuji P.
      • Davidson N.
      • Lester H.A.
      Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by Gβγ subunits and function as heteromultimers.
      ), facilitated by the development of heterologous expression systems such as Xenopus oocytes, and the cloning of the individual cDNAs responsible for the G proteins αi, β, and γ, which implicated Gβγ as the signal transducer of GIRK.
      These technological advances paved the way for the group of Catterall (
      • Herlitze S.
      • Garcia D.E.
      • Mackie K.
      • Hille B.
      • Scheuer T.
      • Catterall W.A.
      Modulation of Ca2+ channels by G-protein βγ subunits.
      ) to determine that the active G protein subunit responsible for Gi/o-coupled GPCRs’ inhibitory action upon N- and P/Q-type voltage-gated calcium channels was Gβγ and not Gα. This finding was echoed by Ikeda (
      • Ikeda S.R.
      Voltage-dependent modulation of N-type calcium channels by G-protein βγ subunits.
      ) for N-type channels in the same issue of Nature. The contemporaneous findings of Harden and Gilman (
      • Boyer J.L.
      • Waldo G.L.
      • Harden T.K.
      βγ-Subunit activation of G-protein-regulated phospholipase C.
      ) that phospholipase Cβ and type II adenylyl cyclase (
      • Tang W.J.
      • Gilman A.G.
      Type-specific regulation of adenylyl cyclase by G protein βγ subunits.
      ) were activated by Gβγ provided more widespread acceptance that Gβγ–effector interactions were a critical aspect of G protein signaling. Many more direct Gβγ-binding effectors would be identified in that period, including the GRK family of kinases (
      • Pitcher J.A.
      • Inglese J.
      • Higgins J.B.
      • Arriza J.L.
      • Casey P.J.
      • Kim C.
      • Benovic J.L.
      • Kwatra M.M.
      • Caron M.G.
      • Lefkowitz R.J.
      Role of βγ subunits of G proteins in targeting the β-adrenergic receptor kinase to membrane-bound receptors.
      ) and phosphoinositide-3 kinase (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits.
      ), the molecular basis for many of which were characterized through identification of key residues on the Gα-binding surface of Gβγ (
      • Ford C.E.
      • Skiba N.P.
      • Bae H.
      • Daaka Y.
      • Reuveny E.
      • Shekter L.R.
      • Rosal R.
      • Weng G.
      • Yang C.S.
      • Iyengar R.
      • Miller R.J.
      • Jan L.Y.
      • Lefkowitz R.J.
      • Hamm H.E.
      Molecular basis for interactions of G protein βγ subunits with effectors.
      ). The widespread acceptance of Gβγ as a critical signaling protein with a multitude of effectors was instrumental to our studies of its role in the presynaptic regulation of neurotransmitter release. The development and characterization of selective agonists and antagonists for cell surface receptors was instrumental to formulating the current model of G protein receptor signaling, including the field of signaling that modulates neurotransmitter release from the presynaptic terminal.

      The historical basis for presynaptic regulation of neurotransmitter release

      Physiological mechanisms of control over neurotransmitter release are essential for the orderly transduction of signals from presynaptic to postsynaptic neurons. Our understanding of these mechanisms has expanded considerably over the past half-century. Presynaptic regulation of transmitter release is vital to the plasticity of a neuron and the signaling network in which it functions. In various studies, the phenomenon of presynaptic inhibition studies in vertebrates started from analysis of sensory inputs to the spinal cord. As early as the 1930s, presynaptic depolarization from sensory stimulation was shown as a mechanism of physiological modulation of these inputs (
      • Gasser H.S.
      • Graham H.T.
      Potentials produced in the spinal cord by stimulation of dorsal roots.
      ). This was later characterized as primary afferent depolarization (PAD) (
      • Barron D.H.
      • Matthews B.H.
      The interpretation of potential changes in the spinal cord.
      ) and first described as a presynaptic mechanism of inhibition by Frank and Fuortes (
      • Frank K.
      • Fuortes M.G.F.
      Presynaptic and postsynaptic inhibition of monosynaptic reflexes.
      ). Eccles et al. (
      • Eccles J.C.
      • Magni F.
      • Willis W.D.
      Depolarization of central terminals of Group I afferent fibres from muscle.
      ,
      • Eccles J.C.
      • Eccles R.M.
      • Magni F.
      Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys.
      ) went on to firmly link the phenomenon of PAD to presynaptic inhibition as well as to demonstrate that higher brain centers use this mechanism to control sensory inflow (
      • Andersen P.
      • Eccles J.C.
      • Sears T.A.
      Presynaptic inhibitory action of cerebral cortex on the spinal cord.
      ). It was also determined by Eccles et al. (
      • Eccles J.C.
      • Schmidt R.
      • Willis W.D.
      Pharmacological studies on presynaptic inhibition.
      ) that this inhibition is mediated by GABA receptors, although the identity of GABA as an amino acid neurotransmitter was only later proven in vertebrates (
      • Johnston G.A.
      • Beart P.M.
      • Curtis D.R.
      • Game C.J.
      • McCulloch R.M.
      • Maclachlan R.M.
      Bicuculline methochloride as a GABA antagonist.
      ) following extensive work in invertebrate models (
      • Florey E.
      • Chapman D.D.
      The non-identity of the transmitter substance of crustacean inhibitory neurons and γ-aminobutyric acid.
      • Bazemore A.
      • Elliott K.A.
      • Florey E.
      Factor I and γ-aminobutyric acid.
      ,
      • Kravitz E.A.
      • Kuffler S.W.
      • Potter D.D.
      γ-Aminobutyric acid and other blocking compounds in crustacea. III. Their relative concentrations in separated motor and inhibitory axons.
      • Kuffler S.W.
      • Edwards C.
      Mechanism of γ aminobutyric acid (GABA) action and its relation to synaptic inhibition.
      ). Ultrastuctural and immunohistochemical data showing GABAergic axo-axonic synapses later reinforced the existence of axo-axonic synapses that mediate presynaptic inhibition (
      • Conradi S.
      • Cullheim S.
      • Gollvik L.
      • Kellerth J.O.
      Electron microscopic observations on the synaptic contacts of group Ia muscle spindle afferents in the cat lumbosacral spinal cord.
      ,
      • Maxwell D.J.
      • Bannatyne B.A.
      Ultrastructure of muscle spindle afferent terminations in lamina VI of the cat spinal cord.
      • Barber R.P.
      • Vaughn J.E.
      • Saito K.
      • McLaughlin B.J.
      • Roberts E.
      GABAergic terminals are presynaptic to primary afferent terminals in the substantia gelatinosa of the rat spinal cord.
      ).
      Supporting evidence for the role of GABA in presynaptic inhibition was provided by studies in the periphery on sympathetic nerve terminals and ganglia in which GABA receptors caused depolarization of sympathetic ganglia neurons. In these neurons, the first example of GPCR-mediated presynaptic inhibition was found in response to noradrenaline (
      • Starke K.
      Influence of extracellular noradrenaline on the stimulation-evoked secretion of noradrenaline from sympathetic nerves: evidence for an α-receptor-mediated feed-back inhibition of noradrenaline release.
      • Starke K.
      Influence of α-receptor stimulants on noradrenaline release.
      ,
      • Enero M.A.
      • Langer S.Z.
      • Rothlin R.P.
      • Stefano F.J.E.
      Role of the α-adrenoceptor in regulating noradrenaline overflow by nerve stimulation.
      • Dubocovich M.L.
      • Langer S.Z.
      Negative feed-back regulation of noradrenaline release by nerve stimulation in the perfused cat's spleen: differences in potency of phenoxybenzamine in blocking the pre- and post-synaptic adrenergic receptors.
      ). Strikingly, however, GABA-mediated inhibition at these terminals was not mediated by the ionotropic GABAA receptor, but rather by a Gαi/o-coupled GPCR (
      • Holz 4th, G.G.
      • Rane S.G.
      • Dunlap K.
      GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels.
      • Dunlap K.
      Functional and pharmacological differences between two types of GABA receptor on embryonic chick sensory neurons.
      ,
      • Scott R.H.
      • Dolphin A.C.
      Regulation of calcium currents by a GTP analogue: potentiation of (−)-baclofen-mediated inhibition.
      • Dolphin A.C.
      • Scott R.H.
      Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (−)-baclofen.
      ), the GABAB receptor (
      • Bowery N.G.
      • Hill D.R.
      • Hudson A.L.
      • Doble A.
      • Middlemiss D.N.
      • Shaw J.
      • Turnbull M.
      (−)-Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor.
      ), which responded to the anti-convulsant (−)-baclofen (
      • Bowery N.G.
      • Hudson A.L.
      γ-Aminobutyric acid reduces the evoked release of [3H]noradrenaline from sympathetic nerve terminals [proceedings].
      ). GABAB receptors are distributed throughout the central nervous system, where they also mediate presynaptic inhibition. Indeed, it is now clear that GABAB receptors, along with many other GPCRs, including but not limited to opioid receptors (
      • Maekawa K.
      • Minami M.
      • Yabuuchi K.
      • Toya T.
      • Katao Y.
      • Hosoi Y.
      • Onogi T.
      • Satoh M.
      In situ hybridization study of μ- and κ-opioid receptor mRNAs in the rat spinal cord and dorsal root ganglia.
      ,
      • Dado R.J.
      • Law P.Y.
      • Loh H.H.
      • Elde R.
      Immunofluorescent identification of a delta (δ)-opioid receptor on primary afferent nerve terminals.
      ), cannabinoid receptors (
      • Hohmann A.G.
      • Briley E.M.
      • Herkenham M.
      Pre- and postsynaptic distribution of cannabinoid and μ opioid receptors in rat spinal cord.
      ), α2 adrenergic receptors (
      • Pan Y.Z.
      • Li D.P.
      • Pan H.L.
      Inhibition of glutamatergic synaptic input to spinal lamina II(o) neurons by presynaptic α2-adrenergic receptors.
      ), and metabotropic glutamate receptors (
      • Chen S.R.
      • Pan H.L.
      Distinct roles of group III metabotropic glutamate receptors in control of nociception and dorsal horn neurons in normal and nerve-injured Rats.
      ), inhibit glutamate release at spinal sensory synapses, but also throughout the central nervous system.
      A critical result in determining a mechanism by which GPCRs mediate presynaptic inhibition came from recordings from dorsal root ganglion cells in culture. These are the cells that give rise to spinal sensory synapses, which demonstrate PAD, thus understanding their cellular biology provides insight into the biochemistry of their presynaptic terminals. Various GPCR agonists (
      • Dunlap K.
      • Fischbach G.D.
      Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones.
      ,
      • Dunlap K.
      • Fischbach G.D.
      Neurotransmitters decrease the calcium component of sensory neurone action potentials.
      ) that target G proteins in dorsal root ganglia cells (
      • Scott R.H.
      • Dolphin A.C.
      Regulation of calcium currents by a GTP analogue: potentiation of (−)-baclofen-mediated inhibition.
      ,
      • Dolphin A.C.
      • Scott R.H.
      Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (−)-baclofen.
      ,
      • Scott R.H.
      • Dolphin A.C.
      Activation of a G protein promotes agonist responses to calcium channel ligands.
      ) directly inhibited Ca2+ currents, including both noradrenaline α2-adrenergic and GABAB receptors. This led to the hypothesis that GPCRs cause presynaptic inhibition by inhibiting presynaptic Ca2+ entry, although this has not been demonstrated directly at spinal sensory synapses. Nevertheless, it is now clear that presynaptic inhibition is ubiquitous and is found throughout the central nervous system (
      • Bowery N.G.
      • Hill D.R.
      • Hudson A.L.
      • Doble A.
      • Middlemiss D.N.
      • Shaw J.
      • Turnbull M.
      (−)-Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA receptor.
      ,
      • Alford S.
      • Christenson J.
      • Grillner S.
      Presynaptic GABAA and GABAB receptor-mediated phasic modulation in axons of spinal motor interneurons.
      ,
      • Alford S.
      • Grillner S.
      The involvement of GABAB receptors and coupled G-proteins in spinal GABAergic presynaptic inhibition.
      • Andrade R.
      • Malenka R.C.
      • Nicoll R.A.
      A G protein couples serotonin and GABAB receptors to the same channels in hippocampus.
      ). Whereas some presynaptic GPCRs clearly inhibit neurotransmission by inhibiting presynaptic Ca2+ entry (
      • Wu L.G.
      • Saggau P.
      GABAB receptor-mediated presynaptic inhibition in guinea-pig hippocampus is caused by reduction of presynaptic Ca2+ influx.
      • Wu L.G.
      • Saggau P.
      Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus.
      ,
      • Hamid E.
      • Church E.
      • Wells C.A.
      • Zurawski Z.
      • Hamm H.E.
      • Alford S.
      Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.
      • Mizutani H.
      • Hori T.
      • Takahashi T.
      5-HT1B receptor-mediated presynaptic inhibition at the calyx of Held of immature rats.
      ), it is also clear that other mechanisms targeting the release machinery directly are also important (
      • Silinsky E.M.
      On the mechanism by which adenosine receptor activation inhibits the release of acetylcholine from motor nerve endings.
      ,
      • Blackmer T.
      • Larsen E.C.
      • Takahashi M.
      • Martin T.F.
      • Alford S.
      • Hamm H.E.
      G protein βγ subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.
      ).

      Identification of the Gβγ–SNARE interaction as a critical inhibitory mechanism of exocytosis downstream of Ca2+ entry

      Many Gi/o-coupled GPCRs were shown to inhibit exocytosis via the action of Gβγ on voltage-gated calcium channels to inhibit Ca2+ fluxes (
      • McCool B.A.
      • Farroni J.S.
      A1 adenosine receptors inhibit multiple voltage-gated Ca2+ channel subtypes in acutely isolated rat basolateral amygdala neurons.
      • Kobayashi S.
      • Conforti L.
      • Zhu W.H.
      • Beitner-Johnson D.
      • Millhorn D.E.
      Role of the D2 dopamine receptor in molecular adaptation to chronic hypoxia in PC12 cells.
      ,
      • Noguchi J.
      • Yamashita H.
      Adenosine inhibits voltage-dependent Ca2+ currents in rat dissociated supraoptic neurones via A1 receptors.
      ,
      • Huang C.C.
      • Lo S.W.
      • Hsu K.S.
      Presynaptic mechanisms underlying cannabinoid inhibition of excitatory synaptic transmission in rat striatal neurons.
      • Kajikawa Y.
      • Saitoh N.
      • Takahashi T.
      GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABA(B) receptor.
      ). Other researchers found that inhibition occurred at a distinct site downstream of Ca2+ entry (
      • Silinsky E.M.
      On the mechanism by which adenosine receptor activation inhibits the release of acetylcholine from motor nerve endings.
      ,
      • Takahashi M.
      • Freed R.
      • Blackmer T.
      • Alford S.
      Calcium influx-independent depression of transmitter release by 5-HT at lamprey spinal cord synapses.
      ,
      • Silinsky E.M.
      • Solsona C.S.
      Calcium currents at motor nerve endings: absence of effects of adenosine receptors agonists in the frog.
      ). The large size of the sea lamprey (Petromyzon marinus) giant axon preparation, utilized by our group as a model of synaptic transmission, facilitated the direct axonal injection of proteins, such as Gβγ, or critical Gβγ-chelating peptides, such as βARK-CT. Injection of Gβγ inhibited EPSCs without perturbing voltage-gated Ca2+ currents, whereas the chelating peptide blocked the inhibitory effect of the lamprey serotonin receptor, a receptor homologous to human 5-HT1B (
      • Blackmer T.
      • Larsen E.C.
      • Takahashi M.
      • Martin T.F.
      • Alford S.
      • Hamm H.E.
      G protein βγ subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.
      ). Later studies expanded upon this initial finding, showing that the step inhibited by Gβγ was late in the vesicular docking and priming cycle and could occur within tens of milliseconds after the uncaging of agonist (
      • Gerachshenko T.
      • Blackmer T.
      • Yoon E.J.
      • Bartleson C.
      • Hamm H.E.
      • Alford S.
      Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition.
      ). In addition, 5-HT action on the lamprey serotonin receptor reduced quantal size and prevented full fusion (
      • Photowala H.
      • Blackmer T.
      • Schwartz E.
      • Hamm H.E.
      • Alford S.
      G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.
      ), reinforcing the notion that Gβγ was acting at a late step and not influencing vesicular pool sizes via this mechanism.
      No data were shown pertaining to the effector target of Gβγ in the original study from 2001 (
      • Blackmer T.
      • Larsen E.C.
      • Takahashi M.
      • Martin T.F.
      • Alford S.
      • Hamm H.E.
      G protein βγ subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.
      ), but in 2005, the presynaptic release machinery was shown to be the target (
      • Gerachshenko T.
      • Blackmer T.
      • Yoon E.J.
      • Bartleson C.
      • Hamm H.E.
      • Alford S.
      Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition.
      ), because botulinum neurotoxin type A (BoNT/A) cleaves the nine C-terminal residues of the peripheral membrane t-SNARE SNAP-25 (
      • Schiavo G.
      • Santucci A.
      • Dasgupta B.R.
      • Mehta P.P.
      • Jontes J.
      • Benfenati F.
      • Wilson M.C.
      • Montecucco C.
      Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.
      ,
      • Binz T.
      • Blasi J.
      • Yamasaki S.
      • Baumeister A.
      • Link E.
      • Südhof T.C.
      • Jahn R.
      • Niemann H.
      Proteolysis of SNAP-25 by types E and A botulinal neurotoxins.
      ), leaving a truncated SNAP-25Δ9 that is still fusogenic (
      • Banerjee A.
      • Kowalchyk J.A.
      • DasGupta B.R.
      • Martin T.F.
      SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis.
      ,
      • Xu T.
      • Binz T.
      • Niemann H.
      • Neher E.
      Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity.
      ), albeit to a greatly reduced extent. BoNT/A completely eliminated any inhibition by 5-HT. This was confirmed in mammalian tissue, by recording effects of 5-HT1B receptors on glutamate release from CA1 pyramidal neurons (
      • Hamid E.
      • Church E.
      • Wells C.A.
      • Zurawski Z.
      • Hamm H.E.
      • Alford S.
      Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.
      ). We also showed direct in vitro binding of Gβγ to the t-SNARE proteins SNAP-25 and syntaxin 1A and synaptobrevin, as well as t-SNARE and ternary SNARE complexes (
      • Blackmer T.
      • Larsen E.C.
      • Bartleson C.
      • Kowalchyk J.A.
      • Yoon E.J.
      • Preininger A.M.
      • Alford S.
      • Hamm H.E.
      • Martin T.F.
      G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis.
      ,
      • Yoon E.J.
      • Gerachshenko T.
      • Spiegelberg B.D.
      • Alford S.
      • Hamm H.E.
      Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
      ). These studies showed further that competition occurred between Gβγ and the synaptic calcium sensor synaptotagmin 1 for binding sites upon SNAP-25. From this, it was hypothesized that Gβγ could displace synaptotagmin 1 at docked and primed SNARE complexes, preventing it from performing its fusogenic lipid-mixing activity. At higher levels of Ca2+, the fully Ca2+-occupied synaptotagmin would bind more tightly to the membrane-bound SNARE complex (
      • Blackmer T.
      • Larsen E.C.
      • Bartleson C.
      • Kowalchyk J.A.
      • Yoon E.J.
      • Preininger A.M.
      • Alford S.
      • Hamm H.E.
      • Martin T.F.
      G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis.
      ,
      • Yoon E.J.
      • Gerachshenko T.
      • Spiegelberg B.D.
      • Alford S.
      • Hamm H.E.
      Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
      • Tucker W.C.
      • Weber T.
      • Chapman E.R.
      Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs.
      ) and be more able to overcome the inhibition of Gβγ (
      • Blackmer T.
      • Larsen E.C.
      • Bartleson C.
      • Kowalchyk J.A.
      • Yoon E.J.
      • Preininger A.M.
      • Alford S.
      • Hamm H.E.
      • Martin T.F.
      G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis.
      ,
      • Yoon E.J.
      • Gerachshenko T.
      • Spiegelberg B.D.
      • Alford S.
      • Hamm H.E.
      Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
      ,
      • Zurawski Z.
      • Page B.
      • Chicka M.C.
      • Brindley R.L.
      • Wells C.A.
      • Preininger A.M.
      • Hyde K.
      • Gilbert J.A.
      • Cruz-Rodriguez O.
      • Currie K.P.M.
      • Chapman E.R.
      • Alford S.
      • Hamm H.E.
      Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
      ).
      Lipid-mixing assays in reconstituted systems with pure components lent critical support to this hypothesis. Gβγ inhibited Ca2+-synaptotagmin and SNARE-dependent lipid mixing in a defined system of v- and t-SNARE–harboring liposomes, in a concentration-dependent manner (
      • Zurawski Z.
      • Page B.
      • Chicka M.C.
      • Brindley R.L.
      • Wells C.A.
      • Preininger A.M.
      • Hyde K.
      • Gilbert J.A.
      • Cruz-Rodriguez O.
      • Currie K.P.M.
      • Chapman E.R.
      • Alford S.
      • Hamm H.E.
      Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
      ). This inhibitory action of Gβγ on lipid mixing was more potent at lower concentrations of synaptotagmin 1 C2AB and was greatly diminished in the complete absence of synaptotagmin 1 C2AB. In addition, Gβγ could displace fluorescent synaptotagmin 1 C2AB from t-SNARE–harboring supported lipid bilayers (
      • Zurawski Z.
      • Page B.
      • Chicka M.C.
      • Brindley R.L.
      • Wells C.A.
      • Preininger A.M.
      • Hyde K.
      • Gilbert J.A.
      • Cruz-Rodriguez O.
      • Currie K.P.M.
      • Chapman E.R.
      • Alford S.
      • Hamm H.E.
      Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
      ). These studies, much like the original GPCR reconstitution experiments of Lefkowitz and Gilman’s groups (
      • Cerione R.A.
      • Sibley D.R.
      • Codina J.
      • Benovic J.L.
      • Winslow J.
      • Neer E.J.
      • Birnbaumer L.
      • Caron M.G.
      • Lefkowitz R.J.
      Reconstitution of a hormone-sensitive adenylate cyclase system: the pure β-adrenergic receptor and guanine nucleotide regulatory protein confer hormone responsiveness on the resolved catalytic unit.
      ,
      • May D.C.
      • Ross E.M.
      • Gilman A.G.
      • Smigel M.D.
      Reconstitution of catecholamine-stimulated adenylate cyclase activity using three purified proteins.
      • Ross E.M.
      • Gilman A.G.
      Reconstitution of catecholamine-sensitive adenylate cyclase activity: interactions of solubilized components with receptor-replete membranes.
      ) published in JBC, show the requirements for a critical GPCR signaling pathway in a defined system.

      The molecular requirements for the Gβγ–SNARE interaction

      As stated above, the initial insight that the formed SNARE complex was the effector of Gβγ downstream of Ca2+ entry came from studies of the action of BoNT/A. After BoNT/A treatment, 5-HT1B receptor–mediated presynaptic inhibition was lost in both lamprey giant synapses (
      • Gerachshenko T.
      • Blackmer T.
      • Yoon E.J.
      • Bartleson C.
      • Hamm H.E.
      • Alford S.
      Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition.
      ) and in outputs from CA1 pyramidal neurons (
      • Hamid E.
      • Church E.
      • Wells C.A.
      • Zurawski Z.
      • Hamm H.E.
      • Alford S.
      Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.
      ). Subsequent binding studies with recombinant SNAP-25Δ9 showed only a partial 1.5-fold loss of affinity for Gβγ relative to full-length SNAP-25, however (
      • Yoon E.J.
      • Gerachshenko T.
      • Spiegelberg B.D.
      • Alford S.
      • Hamm H.E.
      Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
      ), implying that other residues were involved.
      In light of this finding, we utilized a peptide-mapping approach to find residues on SNAP-25 that were instrumental to bind Gβγ and confirmed these studies with scanning Ala mutagenesis (
      • Wells C.A.
      • Zurawski Z.
      • Betke K.M.
      • Yim Y.Y.
      • Hyde K.
      • Rodriguez S.
      • Alford S.
      • Hamm H.E.
      Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.
      ). We found nine residues located in two clusters upon SNAP-25. The first cluster was adjacent to the N terminus, whereas the second cluster was adjacent to the C terminus. Mutation of eight of the nine residues to Ala abolished both the ability of Gβγ to bind SNAP-25 and the ability of the lamprey serotonin receptor to inhibit neurotransmission, creating a key mechanistic linkage between Gβγ binding and Gi/o-coupled GPCR-mediated inhibition of exocytosis (
      • Wells C.A.
      • Zurawski Z.
      • Betke K.M.
      • Yim Y.Y.
      • Hyde K.
      • Rodriguez S.
      • Alford S.
      • Hamm H.E.
      Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.
      ). Despite this, mutation of the two residues highlighted by the peptide mapping screen within the C-terminal nine residues to Ala did not phenocopy the effect of BoNT/A with respect to Gi/o-coupled GPCRs (
      • Zurawski Z.
      • Rodriguez S.
      • Hyde K.
      • Alford S.
      • Hamm H.E.
      Gβγ binds to the extreme C terminus of SNAP25 to mediate the action of Gi/o-coupled G protein–coupled receptors.
      ), implying that more of the C terminus was involved. Screening of truncated SNAP-25 constructs for Gβγ binding showed that the extreme C-terminal three residues were involved in the mechanism (
      • Zurawski Z.
      • Rodriguez S.
      • Hyde K.
      • Alford S.
      • Hamm H.E.
      Gβγ binds to the extreme C terminus of SNAP25 to mediate the action of Gi/o-coupled G protein–coupled receptors.
      ). Given the two glycine residues within the extreme C terminus, we speculated that the high flexibility of this region may be required to stabilize Gβγ binding.
      The binding of Gβγ to SNARE may be splice variant-dependent; the two splice variants of SNAP-25, SNAP-25A and SNAP-25B, differ by 9 residues via differential splicing of exon 5 within the palmitoylation domain of the linker region between the two SNARE-forming helices (
      • Bark I.C.
      • Wilson M.C.
      Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25.
      ,
      • Bark I.C.
      Structure of the chicken gene for SNAP-25 reveals duplicated exons encoding distinct isoforms of the protein.
      • Bark I.C.
      • Hahn K.M.
      • Ryabinin A.E.
      • Wilson M.C.
      Differential expression of SNAP-25 protein isoforms during divergent vesicle fusion events of neural development.
      ). The two splice variants exhibit time-dependent expression, with SNAP-25A predominating in embryonic development and early postnatal life and SNAP-25B being the predominant isoform in adults, although SNAP-25a expression is retained in the adult hypothalamus and cortex (
      • Bark I.C.
      • Hahn K.M.
      • Ryabinin A.E.
      • Wilson M.C.
      Differential expression of SNAP-25 protein isoforms during divergent vesicle fusion events of neural development.
      ,
      • Bark C.
      • Bellinger F.P.
      • Kaushal A.
      • Mathews J.R.
      • Partridge L.D.
      • Wilson M.C.
      Developmentally regulated switch in alternatively spliced SNAP-25 isoforms alters facilitation of synaptic transmission.
      • Prescott G.R.
      • Chamberlain L.H.
      Regional and developmental brain expression patterns of SNAP25 splice variants.
      ). These two splice variants appear to differ in their ability to interact with certain Gβγ subunits. Co-immunoprecipitation studies show that genetic ablation of SNAP-25B expression partially reduces the ability to co-immunoprecipitate Gβ2, but not Gβ1, with SNAP-25 in the hippocampus (
      • Daraio T.
      • Valladolid-Acebes I.
      • Brismar K.
      • Bark C.
      SNAP-25a and SNAP-25b differently mediate interactions with Munc18–1 and Gβγ subunits.
      ). The region of SNAP-25 where the two splice variants differ is adjacent to only one of the nine residues critical for Gβγ binding, residue Glu-62 (
      • Wells C.A.
      • Zurawski Z.
      • Betke K.M.
      • Yim Y.Y.
      • Hyde K.
      • Rodriguez S.
      • Alford S.
      • Hamm H.E.
      Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.
      ). Scanning Ala mutagenesis showed no reduction in Gβγ binding after mutagenizing any of the residues that differ between each splice variant. Two possible explanations could account for this phenomenon: first, that this differential binding is due to perturbations in the local environment adjacent to residue Glu-62 and, second, that isoform-dependent palmitoylation results in more efficient localization of SNAP-25 to the G-protein/GPCR complex within the active zone.

      Differential Gi/o-signaling mechanisms in secretory cells: synergy between Gβγ’s action on VGCC and SNARE

      Subsequent to the initial discovery of the Gβγ–SNARE mechanism in the lamprey giant synapse, numerous studies have demonstrated its applicability to several families of Gi/o-coupled GPCRs in a variety of mammalian secretory cells. Over the past 18 years of Gβγ–SNARE research, the development of pharmacological and genetic tools for dissection of presynaptic GPCR mechanisms has allowed researchers to demonstrate its importance in an increasingly sophisticated manner. The release of GABA from Purkinje cells in the cerebellum was shown to occur via two mechanisms, including a spontaneous and cAMP-dependent form of release that was inhibited by group II metabotropic glutamate receptors (mGluRs) under conditions where Ca2+ entry from the extracellular space and internal release are both blocked (
      • Glitsch M.
      Selective inhibition of spontaneous but not Ca2+-dependent release machinery by presynaptic group II mGluRs in rat cerebellar slices.
      ).
      The discovery that the Gβγ–SNARE mechanism could be bypassed via treatment with BoNT/A or the delivery of the SNAP-25 193–206 C-terminal peptide (
      • Gerachshenko T.
      • Blackmer T.
      • Yoon E.J.
      • Bartleson C.
      • Hamm H.E.
      • Alford S.
      Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition.
      ) was critical for distinguishing this mechanism from other pathways that regulate presynaptic release. Biochemical studies showed that the SNAP-25Δ9 produced by BoNT/A had lower affinity for Gβγ than full-length SNAP-25 (
      • Yoon E.J.
      • Gerachshenko T.
      • Spiegelberg B.D.
      • Alford S.
      • Hamm H.E.
      Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
      ). Single fiber inputs from the nociceptive pontine parabrachial nucleus form synapses upon the neurons of the central amygdala, and neurotransmission at this synapse is regulated presynaptically by norepinephrine acting on the α2-adrenergic receptor (
      • Delaney A.J.
      • Crane J.W.
      • Sah P.
      Noradrenaline modulates transmission at a central synapse by a presynaptic mechanism.
      ). The action of this receptor was abolished after treatment with BoNT/A, phenocopying that of the lamprey serotonin receptor. In the paraventricular nuclei of the hypothalamus, κ-opioid receptors regulate release from magnocellular neurosecretory cells with a mechanism that the authors attributed to the action of Gβγ on the release machinery; however, intriguingly, quantal size was not affected by κ-opioid receptor agonism, differing from results observed with the lamprey serotonin receptor (
      • Iremonger K.J.
      • Bains J.S.
      Retrograde opioid signaling regulates glutamatergic transmission in the hypothalamus.
      ). Moreover, this κ-opioid receptor agonism was abolished via treatment with ionomycin to permeabilize the plasma membrane to 2.5 mm Ca2+.
      These latter results using ionomycin are consistent with effects in synapses, where high presynaptic Ca2+ prevents Gβγ-mediated SNARE interactions (
      • Hamid E.
      • Church E.
      • Wells C.A.
      • Zurawski Z.
      • Hamm H.E.
      • Alford S.
      Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.
      ,
      • Gerachshenko T.
      • Blackmer T.
      • Yoon E.J.
      • Bartleson C.
      • Hamm H.E.
      • Alford S.
      Gβγ acts at the C terminus of SNAP-25 to mediate presynaptic inhibition.
      ), but create an intriguing contrast to results observed with amperometry in chromaffin cells, in which μ-opioid agonism remains inhibitory to release subsequent to treatment with ionomycin in 5 mm Ca2+ (
      • Yoon E.J.
      • Hamm H.E.
      • Currie K.P.M.
      G protein βγ subunits modulate the number and nature of exocytotic fusion events in adrenal chromaffin cells independent of calcium entry.
      ). Nevertheless, the charge of the chromaffin cell amperometric spike, a measure homologous to quantal size in neurons, was reduced by overexpression of Gβγ, as was quantal size itself by the action of serotonin receptors in the lamprey giant synapse (
      • Photowala H.
      • Blackmer T.
      • Schwartz E.
      • Hamm H.E.
      • Alford S.
      G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.
      ). In peripheral tissue, the Gβγ–SNARE mechanism also inhibits release. This was first demonstrated to occur in the β cells of the islets of Langerhans, where the α2 adrenergic receptor inhibited insulin release in a manner that could be overcome by BoNT/A or via intracellular chelation of Gβγ with the SNAP-25 193–206 peptide (
      • Zhao Y.
      • Fang Q.
      • Straub S.G.
      • Lindau M.
      • Sharp G.W.
      Noradrenaline inhibits exocytosis via the G protein βγ subunit and refilling of the readily releasable granule pool via the αi1/2 subunit.
      ).
      In the hippocampus at CA1-subicular synapses, the 5-HT1B receptor was shown to inhibit exocytotic release without altering Ca2+ fluxes. Destruction of the exocytotic apparatus with BoNT/C was shown to liberate Gβγ from its site on SNARE; Gβγ is then capable of modulating Ca2+ entry through VDCC (
      • Hamid E.
      • Church E.
      • Wells C.A.
      • Zurawski Z.
      • Hamm H.E.
      • Alford S.
      Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.
      ). GABAB receptors on the same presynaptic terminals acted to reduce Ca2+ fluxes (Fig. 1). Also, in the hippocampus, long-term depression of synaptic strength in presynaptic Schaffer collateral terminals, as well as presynaptic inhibition mediated by group II mGluRs, was similarly shown to be dependent upon Gβγ and the C terminus of SNAP-25 (
      • Zhang X.-L.
      • Upreti C.
      • Stanton P.K.
      Gβγ and the C terminus of SNAP-25 are necessary for long-term depression of transmitter release.
      ).
      Figure thumbnail gr1
      Figure 1Gβγ–SNARE and Gβγ–VGCC act synergistically to inhibit vesicle release. Serotonin (blue) from paracrine sources acts on inhibitory presynaptic 5-HT1B receptors (blue) to liberate Gβγ to bind SNAREs, displacing the fusogenic calcium sensor synaptotagmin I and inhibiting the release of glutamate (green) This mechanism is distal to and synergistic with GABAB heteroreceptors, which in the presence of axo-axonic GABA (red), inhibit calcium fluxes into the terminal (
      • Wu L.G.
      • Saggau P.
      Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus.
      ). Class 2/3 mGluRs, acting as autoreceptors, may also signal via Gβγ–SNARE, as has been demonstrated to occur at the synapse between cone photoreceptors and horizontal cells of the inner nuclear layer of the retina (
      • Van Hook M.J.
      • Babai N.
      • Zurawski Z.
      • Yim Y.Y.
      • Hamm H.E.
      • Thoreson W.B.
      A presynaptic group III mGluR recruits Gβγ/SNARE interactions to inhibit synaptic transmission by cone photoreceptors in the vertebrate retina.
      ).
      From this group of studies as a whole, we hypothesize that Gβγ–SNARE and Gβγ–VGCC actions are synergistic, receptor-specific mechanisms to provide more complete temporal and scalar control over vesicular release. In hippocampal CA1-subicular paired recordings, greater extent of inhibition was observed by treating with both a 5-HT1B agonist and a GABAB agonist (Fig. 2). The addition of a GABAB agonist prevents recovery of EPSC amplitude subsequent to treatment with the 5-HT1B agonist during repetitive stimulation. We interpret these data as the GABAB agonist blocking the influx of Ca2+ into the terminal, preventing Ca2+ from binding to synaptotagmin and thus reducing its competition with Gβγ on the SNARE complex and allowing augmented inhibition. This paradigm of Gi/o-coupled GPCRs working in tandem to synergistically inhibit release may be present for a multitude of receptor systems at many different synapses.
      Figure thumbnail gr2
      Figure 2Synergy between presynaptic 5-HT1B and GABAB receptors at the CA1-subicular synapse. A, schematic of recording paradigm within mouse hippocampus for CA1-subicular synapses. At these terminals, 5-HT1B receptors release Gβγ to bind SNAREs, and GABAB receptors release Gβγ to inhibit Ca2+ channels. B, stimulation of the CA1-subicular pathway evoked whole-cell recorded EPSCs in subicular pyramidal neurons. During repetitive stimulation, CP93129 (400 nm; blue) substantially inhibited the first response, but in subsequent responses, amplitudes recovered almost 5-fold to their original height. The ratio of inhibition of the first versus the fifth response was 4.6 ± 0.8. C, baclofen (1 μm; green) uniformly inhibited EPSCs throughout the stimulus train. The addition of CP93129 in tandem with baclofen (pink) substantially inhibited responses throughout the stimulus train to a greater extent than baclofen alone (***, p = 0.0002). D, bar graph showing the ratio of the amplitudes of the first and fifth stimulation of the effects of CP93129 alone and after the addition of baclofen (1 μm) (
      • Zurawski Z.
      • Thompson Gray A.D.
      • Brady L.J.
      • Page B.
      • Church E.
      • Harris N.A.
      • Dohn M.R.
      • Yim Y.Y.
      • Hyde K.
      • Mortlock D.P.
      • Winder D.G.
      • Alford S.
      • Jones C.K.
      • Hamm H.E.
      Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes.
      ). Error bars represent mean ± S. E. Adapted from Ref.
      • Zurawski Z.
      • Thompson Gray A.D.
      • Brady L.J.
      • Page B.
      • Church E.
      • Harris N.A.
      • Dohn M.R.
      • Yim Y.Y.
      • Hyde K.
      • Mortlock D.P.
      • Winder D.G.
      • Alford S.
      • Jones C.K.
      • Hamm H.E.
      Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes.
      . This research was originally published in Science Signaling. Zurawski et al. Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes. Science Signaling 2019. ©American Association for the Advancement of Science.

      The development of mouse genetic models deficient in the Gβγ–SNARE interaction

      The idea of Gβγ–SNARE–dependent GPCRs and Gβγ–VGCC–dependent GPCRs acting in a synergistic manner was expanded upon with the development of the SNAP-25Δ3 mouse model, which carries a point mutation in SNAP-25 (Gly-204*), truncating it by three residues. The mutation partially disables the inhibitory Gβγ–SNARE mechanism (
      • Zurawski Z.
      • Rodriguez S.
      • Hyde K.
      • Alford S.
      • Hamm H.E.
      Gβγ binds to the extreme C terminus of SNAP25 to mediate the action of Gi/o-coupled G protein–coupled receptors.
      ,
      • Zurawski Z.
      • Thompson Gray A.D.
      • Brady L.J.
      • Page B.
      • Church E.
      • Harris N.A.
      • Dohn M.R.
      • Yim Y.Y.
      • Hyde K.
      • Mortlock D.P.
      • Winder D.G.
      • Alford S.
      • Jones C.K.
      • Hamm H.E.
      Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes.
      ) while maintaining full exocytotic activity. This mouse is viable in the homozygous state, enabling researchers to assess the importance of the Gβγ–SNARE mechanism in all SNAP-25–expressing secretory cell populations. Critically, the model permits researchers to investigate the mechanism in the absence of toxic reagents, such as BoNT/A or Cd2+, that could render behavioral or physiological studies impossible. Field potential studies at the CA1-subicular synapse in the SNAP-25Δ3 homozygote phenocopy the results (
      • Zurawski Z.
      • Thompson Gray A.D.
      • Brady L.J.
      • Page B.
      • Church E.
      • Harris N.A.
      • Dohn M.R.
      • Yim Y.Y.
      • Hyde K.
      • Mortlock D.P.
      • Winder D.G.
      • Alford S.
      • Jones C.K.
      • Hamm H.E.
      Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes.
      ) obtained with BoNT/A in WT animals (
      • Hamid E.
      • Church E.
      • Wells C.A.
      • Zurawski Z.
      • Hamm H.E.
      • Alford S.
      Modulation of neurotransmission by GPCRs is dependent upon the microarchitecture of the primed vesicle complex.
      ), indicating a role for Gβγ–SNARE in the 5-HT1B receptor, but not the GABAB receptor.
      The animal model was also used to identify a new role for the Gβγ–SNARE mechanism on attenuating stress in the bed nuclei of the stria terminals (BNST), where presynaptic inhibition upon excitatory parabrachial inputs onto BNST neurons was shown to occur via the α2A adrenergic receptor, but not the GABAB receptor. Moving forward, we anticipate that the SNAP-25Δ3 model will be an important tool for investigators to assess the role of the Gβγ–SNARE mechanism in vivo. This animal model will be invaluable to assess chronic, whole-organism effects of Gβγ–SNARE disruption. In the future, we also plan to develop genetic models that permit tissue-specific ablation of the mechanism to more directly assess its importance in individual circuits. This would allow researchers the ability to identify specific neuronal subtypes where Gβγ–SNARE could be a key regulatory mechanism.

      Specificity of Gβγ signaling

      A growing body of work supports the notion of subunit specificity for a subset of effectors of the five known Gβ and 12 Gγ subunits, most of which can heterodimerize with each other in vitro, although in vivo evidence is more scarce (
      • Gautam N.
      • Downes G.B.
      • Yan K.
      • Kisselev O.
      The G-protein βγ complex.
      ,
      • Khan S.M.
      • Sleno R.
      • Gora S.
      • Zylbergold P.
      • Laverdure J.P.
      • Labbé J.C.
      • Miller G.J.
      • Hébert T.E.
      The expanding roles of Gβγ subunits in G protein-coupled receptor signaling and drug action.
      ). Genetic studies show that despite considerable homology, Gβ and Gγ subunits perform specific roles in development. For example, genetic ablation of Gβ1 leads to microencephaly or neural tube closure deficits: homozygous null mutants died within 2 days of birth (
      • Okae H.
      • Iwakura Y.
      Neural tube defects and impaired neural progenitor cell proliferation in Gβ1-deficient mice.
      ). Correspondingly, human de novo loss-of-function mutations in Gβ1 were associated with hypotonia and seizures along with prominent neurodevelopmental disability (
      • Petrovski S.
      • Küry S.
      • Myers C.T.
      • Anyane-Yeboa K.
      • Cogné B.
      • Bialer M.
      • Xia F.
      • Hemati P.
      • Riviello J.
      • Mehaffey M.
      • Besnard T.
      • Becraft E.
      • Wadley A.
      • Politi A.R.
      • Colombo S.
      • et al.
      Germline de novo Mutations in GNB1 cause severe neurodevelopmental disability, hypotonia, and seizures.
      ). Gβ2 is a mediator of neuronal excitability through inhibition of T-type Ca2+ channels (
      • Wolfe J.T.
      • Wang H.
      • Howard J.
      • Garrison J.C.
      • Barrett P.Q.
      T-type calcium channel regulation by specific G-protein βγ subunits.
      ,
      • DePuy S.D.
      • Yao J.
      • Hu C.
      • McIntire W.
      • Bidaud I.
      • Lory P.
      • Rastinejad F.
      • Gonzalez C.
      • Garrison J.C.
      • Barrett P.Q.
      The molecular basis for T-type Ca2+ channel inhibition by G protein β2γ2 subunits.
      ).
      5, which shows the least homology to the other Gβ subunits, has a unique capacity to form heterodimers with the Gγ-like domain of the regulator of G protein signaling (RGS) proteins, such as RGS9, in addition to several studies showing heterodimer formation with Gγ2 (
      • Yost E.A.
      • Mervine S.M.
      • Sabo J.L.
      • Hynes T.R.
      • Berlot C.H.
      Live cell analysis of G protein β5 complex formation, function, and targeting.
      ,
      • Dingus J.
      • Wells C.A.
      • Campbell L.
      • Cleator J.H.
      • Robinson K.
      • Hildebrandt J.D.
      G protein βγ dimer formation: Gβ and Gγ differentially determine efficiency of in vitro dimer formation.
      • Zhang S.
      • Coso O.A.
      • Lee C.
      • Gutkind J.S.
      • Simonds W.F.
      Selective activation of effector pathways by brain-specific G protein β5.
      ). Genetic ablation of Gβ5 shows a very different pattern of phenotypes to Gβ1; Gβ5 knockout mice showed growth deficits, abnormal motor behavior (“tiptoe-walking” in the unstressed state, and hyperactivity (
      • Zhang J.H.
      • Pandey M.
      • Seigneur E.M.
      • Panicker L.M.
      • Koo L.
      • Schwartz O.M.
      • Chen W.
      • Chen C.K.
      • Simonds W.F.
      Knockout of G protein β5 impairs brain development and causes multiple neurologic abnormalities in mice.
      ,
      • Chen C.K.
      • Eversole-Cire P.
      • Zhang H.
      • Mancino V.
      • Chen Y.J.
      • He W.
      • Wensel T.G.
      • Simon M.I.
      Instability of GGL domain-containing RGS proteins in mice lacking the G protein β-subunit Gβ5.
      ), despite being viable. Gγ3-null mice exhibit handling-induced seizures with associated mortality, while being less susceptible to diet-induced obesity than WT littermates, whereas Gγ7-null mice have deficits in adenylyl cyclase activity and increased startle response, but normal locomotion (
      • Schwindinger W.F.
      • Betz K.S.
      • Giger K.E.
      • Sabol A.
      • Bronson S.K.
      • Robishaw J.D.
      Loss of G protein γ7 alters behavior and reduces striatal αolf level and cAMP production.
      ,
      • Schwindinger W.F.
      • Giger K.E.
      • Betz K.S.
      • Stauffer A.M.
      • Sunderlin E.M.
      • Sim-Selley L.J.
      • Selley D.E.
      • Bronson S.K.
      • Robishaw J.D.
      Mice with deficiency of G protein γ3 are lean and have seizures.
      ). These variable phenotypes resulting from genetic ablation of individual Gβ and Gγ subunits suggest isoform dependence for Gβγ dimerization and specificity in signaling roles (
      • Albert P.R.
      • Robillard L.
      G protein specificity: traffic direction required.
      ,
      • Lim W.K.
      • Myung C.-S.
      • Garrison J.C.
      • Neubig R.R.
      Receptor-G protein γ specificity: γ11 shows unique potency for A1 adenosine and 5-HT1A receptors.
      • Lindorfer M.A.
      • Myung C.-S.
      • Savino Y.
      • Yasuda H.
      • Khazan R.
      • Garrison J.C.
      Differential activity of the G protein β5γ2 subunit at receptors and effectors.
      ). Multiple independent groups have observed heterodimer or isoform-dependent coupling of receptors to effectors (
      • Betke K.M.
      • Wells C.A.
      • Hamm H.E.
      GPCR mediated regulation of synaptic transmission.
      ,
      • Robishaw J.D.
      • Berlot C.H.
      Translating G protein subunit diversity into functional specificity.
      ). This may be attributable to variations in expression level, localization, or affinity for effectors within each Gβ and Gγ subunit (
      • Yim Y.Y.
      • McDonald W.H.
      • Hyde K.
      • Cruz-Rodríguez O.
      • Tesmer J.J.G.
      • Hamm H.E.
      Quantitative multiple-reaction monitoring proteomic analysis of Gβ and Gγ subunits in C57Bl6/J brain synaptosomes.
      ); for example, somatostatin receptors in RINm5F cells were specifically shown to inhibit VGCC via β1γ3 (
      • Yim Y.Y.
      • McDonald W.H.
      • Hyde K.
      • Cruz-Rodríguez O.
      • Tesmer J.J.G.
      • Hamm H.E.
      Quantitative multiple-reaction monitoring proteomic analysis of Gβ and Gγ subunits in C57Bl6/J brain synaptosomes.
      • Smrcka A.V.
      G protein βγ subunits: central mediators of G protein-coupled receptor signaling.
      ,
      • Degtiar V.E.
      • Wittig B.
      • Schultz G.
      • Kalkbrenner F.
      A specific Go heterotrimer couples somatostatin receptors to voltage-gated calcium channels in RINm5F cells.
      • Hildebrandt J.D.
      Role of subunit diversity in signaling by heterotrimeric G proteins.
      ). In GH3 cells, a similar result was observed for somatostatin receptors coupling to VGCC with Gγ3, but the muscarinic receptor was shown to couple to VGCC via Gγ4 (
      • Kleuss C.
      • Scherübl H.
      • Hescheler J.
      • Schultz G.
      • Wittig B.
      Selectivity in signal transduction determined by γ subunits of heterotrimeric G proteins.
      ). In addition, Gβγ subunits have distinct expression patterns in the brain (
      • Yim Y.Y.
      • McDonald W.H.
      • Hyde K.
      • Cruz-Rodríguez O.
      • Tesmer J.J.G.
      • Hamm H.E.
      Quantitative multiple-reaction monitoring proteomic analysis of Gβ and Gγ subunits in C57Bl6/J brain synaptosomes.
      ,
      • Betty M.
      • Harnish S.W.
      • Rhodes K.J.
      • Cockett M.I.
      Distribution of heterotrimeric G-protein β and γ subunits in the rat brain.
      ,
      • Vanderbeld B.
      • Kelly G.M.
      New thoughts on the role of the βγ subunit in G-protein signal transduction.
      ).
      Furthermore, unique Gβγ isoforms play specific roles in mediating interactions with both receptors and effectors (
      • Albert P.R.
      • Robillard L.
      G protein specificity: traffic direction required.
      ,
      • Lim W.K.
      • Myung C.-S.
      • Garrison J.C.
      • Neubig R.R.
      Receptor-G protein γ specificity: γ11 shows unique potency for A1 adenosine and 5-HT1A receptors.
      • Lindorfer M.A.
      • Myung C.-S.
      • Savino Y.
      • Yasuda H.
      • Khazan R.
      • Garrison J.C.
      Differential activity of the G protein β5γ2 subunit at receptors and effectors.
      ,
      • Robishaw J.D.
      • Berlot C.H.
      Translating G protein subunit diversity into functional specificity.
      ), and isoforms exhibit tissue-dependent specificity for individual receptors or effectors (
      • Schwindinger W.F.
      • Giger K.E.
      • Betz K.S.
      • Stauffer A.M.
      • Sunderlin E.M.
      • Sim-Selley L.J.
      • Selley D.E.
      • Bronson S.K.
      • Robishaw J.D.
      Mice with deficiency of G protein γ3 are lean and have seizures.
      ,
      • Pronin A.N.
      • Gautam N.
      Interaction between G-protein β and γ subunit types is selective.
      ,
      • Schwindinger W.F.
      • Mihalcik L.J.M.
      • Giger K.E.
      • Betz K.S.
      • Stauffer A.M.
      • Linden J.
      • Herve D.
      • Robishaw J.D.
      Adenosine A2A receptor signaling and Golf assembly show a specific requirement for the γ7 subtype in the striatum.
      • Schwindinger W.F.
      • Mirshahi U.L.
      • Baylor K.A.
      • Sheridan K.M.
      • Stauffer A.M.
      • Usefof S.
      • Stecker M.M.
      • Mirshahi T.
      • Robishaw J.D.
      Synergistic roles for G-protein γ3 and γ7 subtypes in seizure susceptibility as revealed in double knockout mice.
      ). This has been observed for the t-SNARE complex, where Gβ1γ2 has a 14-fold higher affinity than Gβ1γ1 (
      • Zurawski Z.
      • Page B.
      • Chicka M.C.
      • Brindley R.L.
      • Wells C.A.
      • Preininger A.M.
      • Hyde K.
      • Gilbert J.A.
      • Cruz-Rodriguez O.
      • Currie K.P.M.
      • Chapman E.R.
      • Alford S.
      • Hamm H.E.
      Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
      ) and a 20-fold higher potency at inhibiting exocytosis (
      • Blackmer T.
      • Larsen E.C.
      • Bartleson C.
      • Kowalchyk J.A.
      • Yoon E.J.
      • Preininger A.M.
      • Alford S.
      • Hamm H.E.
      • Martin T.F.
      G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis.
      ). However, the lack of subunit-specific antibodies for each Gβ and Gγ subunit limits the understanding of Gβγ specificity. Using a powerful quantitative proteomic approach (
      • Yim Y.Y.
      • McDonald W.H.
      • Hyde K.
      • Cruz-Rodríguez O.
      • Tesmer J.J.G.
      • Hamm H.E.
      Quantitative multiple-reaction monitoring proteomic analysis of Gβ and Gγ subunits in C57Bl6/J brain synaptosomes.
      ), we are beginning to get a glimpse of the specificity by which different Gβγ subunits are recruited to both GPCRs (
      • Yim Y.Y.
      • McDonald W.H.
      • Hyde K.
      • Cruz-Rodríguez O.
      • Tesmer J.J.G.
      • Hamm H.E.
      Quantitative multiple-reaction monitoring proteomic analysis of Gβ and Gγ subunits in C57Bl6/J brain synaptosomes.
      ) and the SNARE complex (
      • Yim Y.Y.
      • Betke K.M.
      • McDonald W.H.
      • Gilsbach R.
      • Chen Y.
      • Hyde K.
      • Wang Q.
      • Hein L.
      • Hamm H.E.
      The in vivo specificity of synaptic Gβ and Gγ subunits to the α2a adrenergic receptor at CNS sites.
      ). For example, we determined the in vivo Gβγ specificity of presynaptic α2a-adrenergic receptors (α2aARs) in both adrenergic (auto-α2aARs) and nonadrenergic neurons (hetero-α2aARs) (
      • Yim Y.Y.
      • Betke K.M.
      • McDonald W.H.
      • Gilsbach R.
      • Chen Y.
      • Hyde K.
      • Wang Q.
      • Hein L.
      • Hamm H.E.
      The in vivo specificity of synaptic Gβ and Gγ subunits to the α2a adrenergic receptor at CNS sites.
      ). This study suggested that auto- and hetero-α2aARs utilize different Gβγ subunits to regulate their downstream signaling mechanisms. We are currently using similar approaches to delineate mechanisms of Gβγ subunit specificity to SNARE complexes to continue to pursue the remarkable specificity of Gβ and Gγ subunits.

      Future directions

      Negative regulators of signaling of many types are very important for shaping signals. For example, RGS proteins and phosphatases both have very important roles in controlling the turnoff rate of signal transduction. The inhibition of secretion is no different. This type of regulation is like a rheostat; it does not affect GPCR activity per se, or signaling through the various G protein α and most βγ subunit pathways, except it blunts SNARE-mediated exocytosis. The Gβγ–SNARE interaction can be a convergent point for various hormones and neuromodulators known to participate in inhibition of exocytosis.
      Although Gα subunit signaling and its dysregulation in diseases has been studied in great detail, understanding how the different Gβγ subunits show as much specificity to receptors as well as effectors will prove to be an important insight. It will be of interest to explore whether and how Gβγ–SNARE interaction plays a role in neural and endocrine disorders.
      We noticed that the phenotypes resulting from disabling Gβγ–SNARE interaction are of variable strength, with, for example, subtle effects on locomotion but dramatic effects on stress responses (
      • Zurawski Z.
      • Thompson Gray A.D.
      • Brady L.J.
      • Page B.
      • Church E.
      • Harris N.A.
      • Dohn M.R.
      • Yim Y.Y.
      • Hyde K.
      • Mortlock D.P.
      • Winder D.G.
      • Alford S.
      • Jones C.K.
      • Hamm H.E.
      Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes.
      ). This leads us to suggest that targeting the Gβγ–SNARE interaction might have some level of specificity. In addition, specificity might be improved by combining an inhibitor with an antagonist of a presynaptic Gi/o-coupled receptor (Fig. 3). The two agents would be on the same pathway, and we hypothesize that untoward side effects of each could be mitigated. A great deal is known about regulation of exocytosis by Gβγ-VDCC interaction, and this has led to a large class of pharmaceuticals that work as calcium channel modulators (
      • Belardetti F.
      • Zamponi G.W.
      Linking calcium-channel isoforms to potential therapies.
      ). The Gβγ–SNARE interaction downstream of calcium entry is much less understood and has been studied in relatively few cases. Enhancement of the release of hormones and neurotransmitters by inhibiting the Gβγ–SNARE interaction could be a novel pharmaceutical strategy. This is an underappreciated locus that has great potential to differentiate various modulatory pathways and to provide fine tuning of hormone or neurotransmitter release within a range of physiological activities. Similarly, regulation of exocytosis through the two Gβγ-mediated modulatory mechanisms enhanced each other’s activity (Fig. 2), leading to a more profound level of inhibition (Fig. 3).
      Figure thumbnail gr3
      Figure 3Potential synergy between GPCR antagonists and Gβγ–SNARE inhibitors. Potential mechanism for a synergistic relationship between existing Gi/o-coupled GPCR antagonists (orange) used clinically to inhibit release presynaptically and hypothetical to-be-developed inhibitors of the Gβγ–SNARE interaction (yellow). The usage of two different inhibitors acting on different targets within the same pathway should more potently drive release than either drug used alone and may permit lower concentrations of antagonist to be utilized, minimizing potential side effects from specificity.

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