Gβγ and the C Terminus of SNAP-25 Are Necessary for Long-Term Depression of Transmitter Release

Abstract

BackgroundShort-term presynaptic inhibition mediated by G protein-coupled receptors involves a direct interaction between G proteins and the vesicle release machinery. Recent studies implicate the C terminus of the vesicle-associated protein SNAP-25 as a molecular binding target of Gβγ that transiently reduces vesicular release. However, it is not known whether SNAP-25 is a target for molecular modifications expressing long-term changes in transmitter release probability.Methodology/Principal FindingsThis study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca2+] increases, in single Schaffer collateral presynaptic release sites in in vitro hippocampal slices, plus simultaneous recording of Schaffer collateral-evoked synaptic potentials. We used electroporation to infuse small peptides through CA3 cell bodies into presynaptic Schaffer collateral terminals to selectively study the presynaptic effect of scavenging the G-protein Gβγ. We demonstrate here that the C terminus of SNAP-25 is necessary for expression of LTD, but not long-term potentiation (LTP), of synaptic strength. Using type A botulinum toxin (BoNT/A) to enzymatically cleave the 9 amino acid C-terminus of SNAP-25 eliminated the ability of low frequency synaptic stimulation to induce LTD, but not LTP, even if release probability was restored to pre-BoNT/A levels by elevating extracellular [Ca2+]. Presynaptic electroporation infusion of the 14-amino acid C-terminus of SNAP-25 (Ct-SNAP-25), to scavenge Gβγ, reduced both the transient presynaptic inhibition produced by the group II metabotropic glutamate receptor stimulation, and LTD. Furthermore, presynaptic infusion of mSIRK, a second, structurally distinct Gβγ scavenging peptide, also blocked the induction of LTD. While Gβγ binds directly to and inhibit voltage-dependent Ca2+ channels, imaging of presynaptic [Ca2+] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca2+ influx, an effect not altered by infusion of Ct-SNAP-25.Conclusions/SignificanceThe C-terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, is a binding target for Gβγ necessary for both transient transmitter-mediated presynaptic inhibition, and the induction of presynaptic LTD.

Gβγ Inhibits Exocytosis via Interaction with Critical Residues on SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein-25

“…In addition to binding to SNAP-25, Yoon et al (2007) showed that G␤␥ also binds individually to the other SNARE proteins, syntaxin 1A and synaptobrevin, as well as to the t-SNARE dimer (SNAP-25 with syntaxin 1A) and to the ternary SNARE complex. This mechanism of G␤␥-mediated inhibition of secretion has been identified in neurons (Blackmer et al, 2001;Delaney et al, 2007;Zhang et al, 2011), chromaffin cells (Yoon et al, 2008), and pancreatic ␤-cells (Zhao et al, 2010).…”

“…In the presynapse, G␤␥ has been shown to be an important regulator of neurotransmission through interactions with calcium channels (Hille, 1994;Ikeda and Dunlap, 1999;Dolphin, 2003) and with the secretory machinery itself (Blackmer et al, 2001Gerachshenko et al, 2005; for review, see Betke et al, 2012). In particular, G␤␥ binds directly to the ternary SNARE complex (a trimer of SNAP-25, syntaxin 1A, and synaptobrevin), as established in biochemical as well as in vitro assays (Blackmer et al, 2001Gerachshenko et al, 2005;Photowala et al, 2006;Delaney et al, 2007;Yoon et al, 2007Yoon et al, , 2008Zhao et al, 2010;Zhang et al, 2011).…”

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

“…Inhibition of exocytosis at this synapse was shown to be mediated by the a 2 adrenergic receptor via the Gbg-SNARE interaction (Delaney et al, 2007). Other mammalian studies include that by Zhang et al, (2011), where introduction of Gbg-scavenging peptides into CA3 hippocampal terminals blocked group II metabotropic glutamate receptor-mediated presynaptic depression of release, and introduction of BoNT/A into Schaffer collateral CA1 synapses reduced induction of long-term depression. These studies are both heavily reliant upon the introduction of Gbg-scavenging peptides and lightchain botulinum toxins to demonstrate the involvement of the Gbg-SNARE interaction.…”

“…Presynaptic G i/o -coupled GPCRs have been shown to be relevant drug targets for anxiety and schizophrenia (Swanson et al, 2005;Patil et al, 2007), but the mechanisms for these effects are not known. The Gbg-SNARE interaction has been shown to be functionally relevant for a number of presynaptic G i/o -coupled GPCRs (Glitsch, 2006;Delaney et al, 2007;Heinke et al, 2011;Zhang et al, 2011;Betke et al, 2012). To explore these and other potential areas of therapeutic relevance further, a transgenic model deficient in the Gbg-SNARE interaction is required.…”

Effects of botulinum toxin type A facial injection on monoamines and their metabolites in sensory, limbic and motor brain regions in rats

“…It is evident that proteolysis of SNARE proteins alters facilitation and depression in a specific way [48,50]. By this, changes in neurotransmission by BTX may be able to influence clinical phenotypes like depression, sleep and pain.…”