Action of botulinum neurotoxin on acetylcholine release from rat brain synaptosomes: putative internalization of the toxin into synaptosomes

The action of botulinum neurotoxin type C1 on the release of acetylcholine from rat brain synaptosomes was studied by using anti-toxin heavy chain Fab and anti-toxin light chain Fab. The toxin was bound to synaptosomes at 0 degrees C for 10 min, in which [14C]acetylcholine had been accumulated previously. The toxin-binding synaptosomes were pre-incubated at 37 degrees C, and the release of acetylcholine was determined after the synaptosomes had been incubated in 25 mM KCl-incubation medium for 20 min at 37 degrees C. Inhibition of [14C]acetylcholine release from the synaptosomes was observed with increasing pre-incubation time and toxin concentration, and the maximum inhibition was seen after pre-incubation for at least 15 min, which was called the "lag time." The toxin-binding synaptosomes were reacted with anti-toxin heavy chain and anti-toxin light chain Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C. Both Fabs reversed the acetylcholine release inhibition by the toxin. However, when the Fabs were added during the pre-incubation time at 37 degrees C, they showed less restoration with increasing pre-incubation time. The restoration was completely abolished if the Fabs were added to the synaptosomes after the first half of the "lag time." On the other hand, when 125I-labeled toxin-binding synaptosomes were reacted with the Fabs at 0 degrees C for 1.5 min before pre-incubation of the synaptosomes at 37 degrees C, anti-heavy chain Fab removed 125I-toxin from the synaptosomes, but anti-light chain Fab did not. However, if the Fabs were added to toxin-binding synaptosomes during the pre-incubation time at 37 degrees C, the Fabs could not remove 125I-toxin from the synaptosomes, and the synaptosomes retained more labeled toxin with increasing pre-incubation time. These results suggest that there are three distinct steps in the inhibition of acetylcholine release from synaptosomes by botulinum neurotoxin. The first is binding, which is reversible, temperature-independent, and mediated by the heavy chain of the toxin. The second is temperature-dependent internalization, that takes place in the first half of the "lag time," in which both the chains are internalized into synaptosomes. The third is the development of toxicity, which requires the latter half of the "lag time."