Botulinum A and the light chain of tetanus toxins inhibit distinct stages of Mg · ATP‐dependent catecholamine exocytosis from permeabilised chromaffin cells

Abstract

Susceptibilities of Mg · ATP-independent and Mg · ATP-requiring components of catecholamine secretion from digitonin-permeabilised chromaffin cells to inhibition by Clostridial botulinum type A and tetanus toxins were investigated. These toxins are Zn²⁺-dependent proteases which specifically cleave the 25-kDa synaptosomal-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) II, respectively. When applied to permeabilised chromaffin cells they rapidly inhibited secretion in the presence of Mg · ATP but the catecholamine released in the absence of Mg · ATP, thought to represent fusion of primed granules, was not perturbed. The toxins can exert their effects per se in the absence of the nucleotide complex; therefore, Mg · ATP-requiring steps of secretion are implicated as roles for their targets. Primed release was lost rapidly after permeabili-sation of the cells but could be maintained by including Mg · ATP during the incubation before stimulating release with Ca²⁺. This ability of Mg · ATP to maintain primed release was only partially inhibited by botulinum neurotoxin A whereas it was abolished by tetanus toxin, consistent with the distinct substrates for these toxins. This study reveals a component of release within which these proteins are either resistant to cleavage by these toxins or in such a position that degradation can no longer prevent granule fusion. Differences in the steps of release at which these toxins can affect inhibition are also revealed.