Modulation of Neurotransmission by GPCRs Is Dependent upon the Microarchitecture of the Primed Vesicle Complex

Abstract

G i/o -protein-coupled receptors (GPCRs) ubiquitously inhibit neurotransmission, principally via G␤␥, which acts via a number of possible effectors. GPCR effector specificity has traditionally been attributed to G␣, based on G␣'s preferential effector targeting in vitro compared with G␤␥'s promiscuous targeting of various effectors. In synapses, however, G␤␥ clearly targets unique effectors in a receptordependent way to modulate synaptic transmission. It remains unknown whether G␤␥ specificity in vivo is due to specific G␤␥ isoformreceptor associations or to spatial separation of distinct G␤␥ pathways through macromolecular interactions. We thus sought to determine how G␤␥ signaling pathways within axons remain distinct from one another. In rat hippocampal CA1 axons, GABA B receptors (GABA B Rs) inhibit presynaptic Ca 2ϩ entry, and we have now demonstrated that 5-HT 1B receptors (5-HT 1B Rs) liberate G␤␥ to interact with SNARE complex C terminals with no effect on Ca 2ϩ entry. Both GABA B Rs and 5-HT 1B Rs inhibit Ca 2ϩ -evoked neurotransmitter release, but 5-HT 1B Rs have no effect on Sr 2ϩ -evoked release. Sr 2ϩ , unlike Ca 2ϩ , does not cause synaptotagmin to compete with G␤␥ binding to SNARE complexes. 5-HT 1B Rs also fail to inhibit release following cleavage of the C terminus of the SNARE complex protein SNAP-25 with botulinum A toxin. Thus, GABA B Rs and 5-HT 1B Rs both localize to presynaptic terminals, but target distinct effectors. We demonstrate that disruption of SNARE complexes and vesicle priming with botulinum C toxin eliminates this selectivity, allowing 5-HT 1B R inhibition of Ca 2ϩ entry. We conclude that receptor-effector specificity requires a microarchitecture provided by the SNARE complex during vesicle priming.

GβγBinds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein–Coupled Receptors

“…Injection of an exogenous mutant SNAP25 containing these eight residues mutated to Ala with a botulinum toxin E (BoNT/E) resistance site into presynaptic neurons, along with BoNT/E light-chain protease, restores fusion while abrogating the ability of serotonin [5-hydroxytryptamine (5-HT)] to inhibit vesicle release in lamprey reticulospinal axons . Interestingly, data were recently shown supporting the notion that a distinct "microarchitecture" is prevalent at presynaptic 5-HT 1b receptors, predisposing them to this mode of Gbg-driven inhibition, whereas other microarchitectures both within the same synapses and within other types of synapses function through other mechanisms, such as the Gbg-mediated inhibition of calcium influx through voltage-gated calcium channels at the GABA B receptor (Hamid et al, 2014). From this, our current understanding of presynaptic inhibition is that presynaptic G i/o -coupled GPCRs function through a variety of mechanisms, including the direct binding of Gbg to SNAP25.…”

“…One well-studied mechanism is the direct binding of G protein bg subunits to voltage-gated calcium channels, leading to voltage-dependent inhibition of calcium entry (Ikeda, 1996). The ability of G i/o -coupled GPCRs to inhibit exocytosis downstream of voltage-gated calcium channels is well documented in a number of different cell types (Blackmer et al, 2001;Delaney et al, 2007;Yoon et al, 2008;Iremonger and Bains, 2009;Zhao et al, 2010;Hamid et al, 2014). We have previously demonstrated that inhibition can also occur through the direct interaction of Gbg with the soluble N-ethylmaleimide attachment protein receptor (SNARE) protein SNAP25 Gerachshenko et al, 2005;Yoon et al, 2007).…”

A Presynaptic Group III mGluR Recruits Gβγ/SNARE Interactions to Inhibit Synaptic Transmission by Cone Photoreceptors in the Vertebrate Retina

“…Although the tiger salamander SNAP-25 sequence is unknown, this site and the rest of the SNAP-25 C terminus are largely conserved in the Japanese fire belly newt (Cynops pyrrhogaster, GenBank accession #BAE47569.1), another amphibian related to the tiger salamander. The actions of BoNT/A at this site have been shown to inhibit G␤␥-SNAP-25 interactions and G␤␥-mediated reductions in synaptic transmission in other neurons (Gerachshenko et al, Delaney et al, 2007;Hamid et al, 2014). In recordings from tissue treated with BoNT/A (n ϭ 5 cone-HC pairs), L-AP4 application resulted in a nonsignificant 8 Ϯ 4% increase in I Ca (p ϭ 0.14, paired t test; n ϭ 5) and a nonsignificant 7 Ϯ 4% reduction in the EPSC amplitude (p ϭ 0.17, paired t test; n ϭ 5).…”

“…) over, direct introduction of G␤␥ subunits dose-dependently inhibited exocytosis, and this effect was inhibited by BoNT/A pretreatment. G␤␥-SNARE interactions triggered by G i/o -coupled GPCRs have been identified in a handful of other synapses: in mammals, the hippocampal CA1-subicular synapse (Hamid et al, 2014), Schaeffer collateral-CA1 synapses (Zhang et al, 2011), and inputs to the amygdala (Delaney et al, 2007), and in lamprey, reticulospinal axon synapses .…”

“…A role for G␤␥/SNARE interactions in regulating synaptic transmission is well established in several systems Delaney et al, 2007;Zhang et al, 2011;Hamid et al, 2014), yet it is unclear whether this occurs at retinal ribbon synapses with their specialized complement of synaptic proteins and distinct signaling properties. In this study, we leverage a powerful set of approaches, including paired whole-cell recordings, whole-cell capacitance recordings, mEPSC and quantal glutamate transporter current analyses, and proximity ligation assay (PLA) to test whether activation of Group III metabotropic glutamate receptors (mGluRs) recruits G␤␥/SNARE interactions to regulate glutamate release at cone ribbon synapses.…”