title: Gβγ and the C terminus of SNAP-25 are necessary for long-term depression of transmitter release.
abstract: BACKGROUND
  Short-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.
  
  
  RESULTS
  This study utilized two-photon laser scanning microscopy for real-time imaging of action potential-evoked [Ca(2+)] 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 [Ca(2+)]. 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 Ca(2+) channels, imaging of presynaptic [Ca(2+)] with Mg-Green revealed that low-frequency stimulation only transiently reduced presynaptic Ca(2+) influx, an effect not altered by infusion of Ct-SNAP-25.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York, United States of America.
authors.forename: Xiao-lei
authors.initials: XL
authors.lastname: Zhang
authors.forename: Chirag
authors.initials: C
authors.lastname: Upreti
authors.forename: Patric K
authors.initials: PK
authors.lastname: Stanton
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D017774:Long-Term Potentiation / Q000187:drug effects / Q000502:physiology; D036881:Long-Term Synaptic Depression / Q000187:drug effects / Q000502:physiology*; D018613:Microscopy, Confocal / Q000379:methods; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology; D009465:Neuromuscular Agents / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017966:Pyramidal Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D009465:Neuromuscular Agents; D010455:Peptides; D050825:Synaptosomal-Associated Protein 25; D019274:Botulinum Toxins, Type A; D002118:Calcium