Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction

Although vesicle-associated membrane protein (VAMP/synaptobrevin) is essent ial for evoked neurotransmitter release, its role in spontaneous transmitte r release remains uncertain. For instance, many studies show that tetanus t oxin (TeNT), which cleaves VAMP, blocks evoked transmitter release but leav es some spontaneous transmitter release. We used recombinant tetanus and bo tulinum neurotoxin catalytic light chains (TeNT-LC, BoNT/B-LC, and BoNT/D-L C) to examine the role of VAMP in spontaneous transmitter release at neurom uscular junctions (nmj) of crayfish. Injection of TeNT-LC into presynaptic axons removed most of the VAMP immunoreactivity and blocked evoked transmit ter release without affecting nerve action potentials or Ca2+ influx. The f requency of spontaneous transmitter release was little affected by the TeNT -LC when the evoked transmitter release had been blocked by >95%. The spont aneous transmitter release left after TeNT-LC treatment was insensitive to increases in intracellular Ca2+. BoNT/B-LC, which cleaves VAMP at the same site as TeNT-LC but uses a different binding site, also blocked evoked rele ase but had minimal effect on spontaneous release. However, BoNT/D-LC, whic h cleaves VAMP at a different site from the other two toxins but binds to t he same position on VAMP as TeNT, blocked both evoked and spontaneous trans mitter release at similar rates. The data indicate that different VAMP comp lexes are employed for evoked and spontaneous transmitter release; the VAMP used in spontaneous release is not readily cleaved by TeNT or BoNT/B. Beca use the exocytosis that occurs after the action of TeNT cannot be increased by increased intracellular Ca2+, the final steps in neurotransmitter relea se are Ca2+ independent.