title: Clues to the multi-phasic inhibitory action of botulinum neurotoxins on release of transmitters.
abstract: 1. With the aim of gaining insight into the mechanism of Ca2(+)-dependent secretion, inhibition of transmitter release by botulinum neurotoxins or their fragments was studied at mammalian motor nerve terminals, cerebrocortical synaptosomes and PC-12 cells. 2. Relative to BoNT type A, the feeble neuromuscular paralytic activity of its two chains and the lack of activity observed with a proteolytic fragment, H2L (lacking H1, the C-terminal half of the heavy chain) highlight a requirement of the intact, disulphide-linked dichain protein for efficient targetting (binding/uptake) to peripheral cholinergic nerve endings. 3. In PC-12 cells, the renatured light chain alone proved equally potent as the whole toxin in reducing Ca2(+)-evoked noradrenaline release, when digitonin-permeabilization was used to overcome the uptake barrier. Treatment of BoNT A with 10 mM dithiothreitol, under non-denaturing conditions, was not very effective in reducing its inter-chain disulphide bond(s) and had little influence on the level of inhibition seen. 4. Altering the intra-synaptosomal concentrations of cyclic nucleotides (c-AMP, c-GMP) or protein kinase C activity failed to affect the reduction of Ca2(+)-dependent K(+)-stimulated noradrenaline release caused by BoNT A or B. On the other hand, raising the cytosolic Ca2+ concentration with the ionophore A23187 reversed the inhibitory effect of BoNT A to a greater extent than that of type B, revealing differences in their actions. 5. Whereas BoNT-induced decrease of Ca2(+)-dependent K(+)-evoked release of noradrenaline was unaffected by destruction of the actin-based cytoskeleton in synaptosomes with cytochalasin D, disassembly of microtubules with colchicine, nocodazole or griseofulvin antagonised the intracellular action of type B but not A. It is speculated that BoNT B blocks transmitter release by interfering with the proposed detachment of synaptic vesicles from microtubules. Establishing the precise involvement of tubulin in the toxin's action may provide a valuable clue to the mechanism of neurotransmitter release or its control.
authors.affiliation: Department of Biochemistry, Imperial College, London, U.K.
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
authors.forename: A C
authors.initials: AC
authors.lastname: Ashton
authors.forename: C
authors.initials: C
authors.lastname: McInnes
authors.forename: J D
authors.initials: JD
authors.lastname: Wadsworth
authors.forename: B
authors.initials: B
authors.lastname: Poulain
authors.forename: L
authors.initials: L
authors.lastname: Tauc
authors.forename: C C
authors.initials: CC
authors.lastname: Shone
authors.forename: J
authors.initials: J
authors.lastname: Melling
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000302:isolation & purification / Q000494:pharmacology*; D008322:Mammals / Q000502:physiology*; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000302:isolation & purification / Q000494:pharmacology*; D014179:Neurotransmitter Uptake Inhibitors / Q000494:pharmacology*; D009638:Norepinephrine / Q000378:metabolism; D009435:Synaptic Transmission
chemical_list: D009498:Neurotoxins; D014179:Neurotransmitter Uptake Inhibitors; D001905:Botulinum Toxins; D009638:Norepinephrine