Importance of Two Adjacent C-terminal Sequences of SNAP-25 in Exocytosis from Intact and Permeabilized Chromaffin Cells Revealed by Inhibition with Botulinum Neurotoxins A and E

Abstract

Types A and E botulinum neurotoxin (BoNT) are Zn²⁺-requiring endoproteases 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 vesicles in bovine adrenochromaffin cells was examined by measuring cleavage of SNAP-25 in relation to the levels of Ca²⁺-evoked catecholamine release from cells exposed to BoNT/A or /E, either before or after permeabilization. The dose-dependency of inhibition of exocytosis correlated closely with the extents of SNAP-25 cleavage in cells permeabilized and 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-complete inhibition of exocytosis; however, after their permeabilization a significant level of secretion was recorded upon Ca²⁺-stimulation. Importantly, 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 decreased the BoNT/A-insensitive exocytosis. When permeabilized cells were exposed to either neurotoxin, both blocked MgATP-dependent secretion but only BoNT/E attenuated the energy-independent phase. These distinct inhibitory effects of the two neurotoxins demonstrate that residues 197−205 at the C-terminus of SNAP-25 are absolutely essential for exocytosis from intact cells whereas even after their removal a significant proportion of the exocytotic response can be elicited from permeabilized cells, but this is reliant on amino acids 180−196. Moreover, the latter but not residues 197−205 are implicated in a late, MgATP-independent step of exocytosis, which is blocked by BoNT/E but nonsusceptible to BoNT/A.