kt-5720 and lactacystin

UNC-13 is a highly conserved plasma membrane-associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC-13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion ("priming") at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC-13 in NMJ synaptic boutons is regulated downstream of GalphaS and Galphaq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC-13 synaptic up-regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC-13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC-13, through a mechanism that appears independent of PKC. Our results suggest that proteasome-mediated protein degradation is the primary mechanism regulating DUNC-13 levels at the synapse. Both PLC- and PKA-mediated pathways appear to regulate synaptic levels of DUNC-13 through controlling the rate of proteasome-dependent DUNC-13 degradation. We conclude that the functional abundance of DUNC-13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways.

Topics: Acetylcysteine; Animals; Antibodies; Blotting, Western; Caenorhabditis elegans Proteins; Carbazoles; Carrier Proteins; Colforsin; Cyclic AMP-Dependent Protein Kinases; Cysteine Endopeptidases; Drosophila; Drosophila Proteins; Drug Interactions; Enzyme Inhibitors; Estrenes; GTP-Binding Proteins; Helminth Proteins; Horseradish Peroxidase; Immunohistochemistry; Indoles; Insect Proteins; Maleimides; Microscopy, Confocal; Multienzyme Complexes; Mutation; Neomycin; Neuromuscular Junction; Oligopeptides; Phorbol Esters; Phospholipid Ethers; Presynaptic Terminals; Proteasome Endopeptidase Complex; Pyrroles; Pyrrolidinones; Synapses; Tumor Suppressor Proteins; Type C Phospholipases