Inhibition of transmitter release by botulinum neurotoxin A

1 The contribution of a proteolytic fragment (H 2 L) of botulinum neurotoxin type A (comprised of the amino‐terminal 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 H 2 L or after intracellular injection into Aplysia neurons. 3 The lack of activity was not the result of alteration in the light chain of H 2 L 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 H 2 L inhibited transmitter release in Aplysia . 4 Bath application of H 2 L 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 H 2 L added to the bath, demonstrating that H 2 L is taken up into cholinergic neurons of Aplysia . This uptake is shown to be mediated by the amino‐terminal moiety of heavy chain (H 2 ), because bath application of light chain plus H 2 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 (H 1 ) since intracellular injection of light chain and H 2 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 H 1 (by inference), whilst H 2 contributes to the internalization step.