title: Distinct exocytotic responses of intact and permeabilised chromaffin cells after cleavage of the 25-kDa synaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinum toxin A or B.
abstract: Botulinum neurotoxin (BoNT) types A and B are Zn2+-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocystosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact 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 almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20microM Ca2+ (and to a lesser extent by 1 mM Ba2+) following permeabilisation. Addition 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 secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of 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 prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
authors.forename: G W
authors.initials: GW
authors.lastname: Lawrence
authors.forename: P
authors.initials: P
authors.lastname: Foran
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D001464:Barium / Q000494:pharmacology; D001905:Botulinum Toxins / Q000378:metabolism* / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism; D002417:Cattle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin; D005089:Exocytosis; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25
chemical_list: D002395:Catecholamines; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D001464:Barium; D000255:Adenosine Triphosphate; D001905:Botulinum Toxins; D004072:Digitonin; D002118:Calcium