IMPORTANCE OF 2 ADJACENT C-TERMINAL SEQUENCES OF SNAP-25 IN EXOCYTOSIS FROM INTACT AND PERMEABILIZED CHROMAFFIN CELLS REVEALED BY INHIBITION WITH BOTULINUM NEUROTOXIN-A AND NEUROTOXIN-E

Types A and E botulinum neurotoxin (BoNT) are Zn2+-requiring endoprote ases which cleave nine and twenty-six residues, respectively, from the C-terminus of synaptosomal-associated protein of M(r) 25 kDa (SNAP-25 ). Involvement of SNAP-25 in the exocytosis of large dense-core vesicl es in bovine adrenochromaffin cells was examined by measuring cleavage of SNAP-25 in relation to the levels of Ca2+-evoked catecholamine rel ease from cells exposed to BoNT/A or /E, either before or after permea bilization. The dose-dependency of inhibition of exocytosis correlated closely with the extents of SNAP-25 cleavage in cells permeabilized a nd then treated with BoNT/E. In intact cells exposed to 66 nM BoNT/A, virtually all of the SNAP-25 was truncated, accompanied by a near-comp lete inhibition of exocytosis; however, after their permeabilization a significant level of secretion was recorded upon Ca2+-stimulation. Im portantly, this BoNT/A-resistant release from the permeabilized cells was dramatically lowered by subsequently adding BoNT/E, which further truncated the SNAP-25 fragment (lacking the C-terminal nine residues) that had been produced earlier by BoNT/A. Moreover, anti-SNAP-25 IgG d ecreased the BoNT/A-insensitive exocytosis. When permeabilized cells w ere exposed to either neurotoxin, both blocked MgATP-dependent secreti on but only BoNT/E attenuated the energy-independent phase. These dist inct inhibitory effects of the two neurotoxins demonstrate that residu es 197-205 at the C-terminus of SNAP-25 are absolutely essential for e xocytosis from intact cells whereas even after their removal a signifi cant proportion of the exocytotic response can be elicited from permea bilized cells, but this is reliant on amino acids 180-196. Moreover, t he latter but not residues 197-205 are implicated in a late, MgATP-ind ependent step of exocytosis, which is blocked by BoNT/E but nonsuscept ible to BoNT/A.