DISTINCT EXOCYTOTIC RESPONSES OF INTACT AND PERMEABILIZED CHROMAFFIN CELLS AFTER CLEAVAGE OF THE 25-KDA SYNAPTOSOMAL-ASSOCIATED PROTEIN (SNAP-25) OR SYNAPTOBREVIN BY BOTULINUM TOXIN-A OR TOXIN-B

Botulinum neurotoxin (BoNT) types A and B are Zn2+-requiring endoprote ases which potently block neurotransmitter release by cleavage of a 25 -kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, resp ectively. Synaptobrevin is important for the exocytosis of catecholami nes from dense-core granules and evidence is presented here for the in volvement of SNAP-25 in this process in neuroendocrine cells. The effe cts of BoNT/A and BoNT/B on regulated secretion were compared in intac t bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by Ba2+, by elevated K+ concentrations or by nicotine was prevented by each toxin . A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT /A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almos t the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residu al fraction of secretion persisted that was induced by 20 mu M Ca2+ (a nd to a lesser extent by 1 mM Ba2+) following permeabilisation. Additi on of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba2+-stimulated BoNT/A-resistant s ecretion. Furthermore, synaptobrevin is essential for this response be cause the response was not observed in BoNT/B treated cells. In view o f the ability of BoNT/E to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not preven t the resultant truncated form exhibiting attenuated activity under th e conditions created by permeabilisation. This identification of a nov el component of secretion from permeabilised cells should facilitate i nvestigation of the functional interaction of SNAP-25 with other prote ins involved in regulated exocytosis.