Differences in the multiple step process of inhibition of neurotransmitter release induced by tetanus toxin and botulinum neurotoxins type A and B at Aplysia synapses

In order to gain insights into the steps (binding, uptake, intracellular effect) which differ in the inhibitory actions of tetanus toxin and botulinum neurotoxins types A or B, their temperature dependencies were investigated at identified cholinergic and non-cholinergic synapses in Aplysia. Upon lowering the temperature from 22 degrees C to 10 degrees C, extracellularly applied botulinum neurotoxin type A and B appeared unable to inhibit transmitter release whilst tetanus toxin exhibited a residual activity. Binding of each toxin to the neuronal membrane appeared virtually unaltered following this temperature change. By contrast, the intracellular effects of botulinum neurotoxin type B acid tetanus toxin were strongly attenuated by temperature reduction whereas the inhibitory action of botulinum neurotoxin type A was only moderately reduced. Importantly, this discrepancy relates to the known proteolytic cleavage of different synaptic proteins by these two toxin groups. Since both the binding and intracellular activity of botulinum neurotoxin type A are minimally affected at 10 degrees C, its inability to inhibit neurotransmission at this low temperature when applied extracellularly indicated attenuation of its uptake. Due to the strict temperature dependence of the intracellular action of tetanus toxin and botulinum neurotoxin type B, but not A, an examination of the effects of changes in temperature on the internalization step was facilitated by the use of heterologous mixtures of the toxins' heavy and light chains. At 10 degrees C, heavy chain from tetanus toxin but not from botulinum neurotoxin type B mediated uptake of botulinum neurotoxin type A light chain. Collectively, these results provide evidence that, at least in Aplysia, the uptake mechanism for botulinum neurotoxin types A and B differs from that of tetanus toxin.