Inhibition of transmitter release by botulinum neurotoxin A. Contribution of various fragments to the intoxication process.
1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.
Poulain
B
B
Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette.
Wadsworth
J D
JD
Maisey
E A
EA
Shone
C C
CC
Melling
J
J
Tauc
L
L
Dolly
J O
JO
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001048:Aplysia; D001905:Botulinum Toxins / Q000633:toxicity*; D009498:Neurotoxins / Q000633:toxicity*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010446:Peptide Fragments / Q000633:toxicity*; D009435:Synaptic Transmission
D009498:Neurotoxins; D018377:Neurotransmitter Agents; D010446:Peptide Fragments; D001905:Botulinum Toxins; D000109:Acetylcholine