INHIBITION OF TRANSMITTER RELEASE CORRELATES WITH THE PROTEOLYTIC ACTIVITY OF TETANUS TOXIN AND BOTULINUS TOXIN-A IN INDIVIDUAL CULTURED SYNAPSES OF HIRUDO-MEDICINALIS

We have studied the effects of tetanus toxin and botulinus toxin A on neurotransmitter release in the Retzius-->P-cell synapse of the leech and exploited the unique properties of this system, which allow for co mbined physiological and biochemical analyses in single-cell pairs. Th e sequences of Hirudo medicinalis synaptobrevin and synaptosomal-assoc iated protein of 25 kDa (SNAP-25), deduced by cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homolog s. Whereas Hirudo synaptobrevin is proteolyzed by tetanus toxin, its S NAP-25 isoform is resistant to botulinus toxin A cleavage because of a mino acid substitutions within and around the putative cleavage site. In close correlation, microinjection of tetanus toxin into the presyna ptic neuron produced a block of transmitter release, whereas botulinus toxin A had no effect on synaptic transmission. Subsequent immunoblot ting of single-cell pairs demonstrated directly that the tetanus toxin -mediated block of exocytosis is accompanied by cleavage of synaptobre vin in the injected neuron, resulting in the generation of a detectabl e C-terminal cleavage product. Immunoblotting also confirmed the resis tance of SNAP-25 to botulinus toxin A cleavage in vivo, Using recombin ant proteins, we show that the N-terminal fragment of synaptobrevin re leased by tetanus toxin, but not its C-terminal membrane-anchored clea vage product, participates with syntaxin and SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in Hirudo. Our data demonst rate a direct correlation between the inhibition of transmitter releas e and the ability of the neurotoxin to proteolyze its target protein a nd support the view that SNARE ternary complex formation is an importa nt step leading to synaptic vesicle exocytosis.