BOTULINUM-A AND THE LIGHT-CHAIN OF TETANUS TOXINS INHIBIT DISTINCT STAGES OF MG-CENTER-DOT-ATP-DEPENDENT CATECHOLAMINE EXOCYTOSIS FROM PERMEABILIZED CHROMAFFIN CELLS

Susceptibilities of Mg . ATP-independent and Mg . ATP-requiring compon ents of catecholamine secretion from digitonin-permeabilised chromaffi n cells to inhibition by Clostridial botulinum type A and tetanus toxi ns were investigated. These toxins are Zn2+-dependent proteases which specifically cleave the 25-kDa synaptosomal-associated protein (SNAP-2 5) and vesicle-associated membrane protein (VAMP) II, respectively. Wh en applied to permeabilised chromaffin cells they rapidly inhibited se cretion in the presence of Mg . ATP but the catecholamine released in the absence of Mg . ATP, thought to represent fusion of primed granule s, was not perturbed. The toxins, can exert their effects per se in th e absence of the nucleotide complex; therefore, Mg . ATP-requiring ste ps of secretion are implicated as roles for their targets. Primed rele ase was lost rapidly after permeabilisation of the cells but could be maintained by including Mg . ATP during the incubation before stimulat ing release with Ca2+. This ability of Mg . ATP to maintain primed rel ease was only partially inhibited by botulinum neurotoxin A whereas it was abolished by tetanus toxin, consistent with the distinct substrat es 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 f usion. Differences in the steps of release at which these toxins can a ffect inhibition are also revealed.