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Volume 17, Number 6,
Issue of March 15, 1997
pp. 1898-1910
Copyright ©1997 Society for Neuroscience
Inhibition of Transmitter Release Correlates with the Proteolytic
Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured
Synapses of Hirudo medicinalis
Received Oct. 28, 1996; revised Dec. 19, 1996; accepted Dec. 30, 1996.
Dieter Bruns1,
Silke Engers1,
Clement Yang1,
Rainer Ossig2,
Andreas Jeromin2, and
Reinhard Jahn1, 2
1 The Howard Hughes Medical Institute and
2 Department of Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06510
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 combined physiological and biochemical analyses in single-cell
pairs. The sequences of Hirudo medicinalis synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25), deduced by
cDNA cloning, are 61 and 55% identical, respectively, to their corresponding mammalian homologs. Whereas Hirudo
synaptobrevin is proteolyzed by tetanus toxin, its SNAP-25 isoform is
resistant to botulinus toxin A cleavage because of amino acid
substitutions within and around the putative cleavage site. In close
correlation, microinjection of tetanus toxin into the presynaptic
neuron produced a block of transmitter release, whereas botulinus toxin
A had no effect on synaptic transmission. Subsequent immunoblotting of
single-cell pairs demonstrated directly that the tetanus toxin-mediated block of exocytosis is accompanied by cleavage of synaptobrevin in the
injected neuron, resulting in the generation of a detectable C-terminal
cleavage product. Immunoblotting also confirmed the resistance of
SNAP-25 to botulinus toxin A cleavage in vivo. Using recombinant proteins, we show that the N-terminal fragment of synaptobrevin released by tetanus toxin, but not its C-terminal membrane-anchored cleavage product, participates with syntaxin and
SNAP-25 in synaptic SNAP receptor (SNARE) ternary complex formation in
Hirudo. Our data demonstrate a direct correlation between the inhibition of transmitter release and the ability of the
neurotoxin to proteolyze its target protein and support the view that
SNARE ternary complex formation is an important step leading to
synaptic vesicle exocytosis.
Key words:
transmitter release;
synaptobrevin;
VAMP;
SNAP-25;
SNARE;
tetanus toxin;
botulinus toxin A;
single-cell immunoblotting;
Hirudo medicinalis
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