Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25 - Identification of the minimal essential C-terminal residues

Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by c leavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoN T/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, thro ughout the same interval, with a continued predominance of cleaved SNAP-25- (1-197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNA P-25-(1-206), no restoration of exocytosis ensued even after 3 weeks. To as certain if this failure was because of the persistence of the toxin's prote ase activity, the cells were transfected with BoNT/A-resistant SNAP-25 cons tructs; importantly, exocytosis was rescued. C-terminal truncation of the t oxin-insensitive SNAP-25 revealed that residues 1-201, 1-202, 1-203 afforde d a significant return of exocytosis, unlike shorter forms 1-197, -198, -19 9, or -200; accordingly, mutants M202A or L203A of full-length SNAP-25 resc ued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provid e the prospect of a therapy for botulism.