title: Multiple domains of botulinum neurotoxin contribute to its inhibition of transmitter release in Aplysia neurons.
abstract: The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.
authors.affiliation: Laboratoire de Neurobiologie Cellulaire et Moléculaire, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
authors.forename: B
authors.initials: B
authors.lastname: Poulain
authors.forename: J D
authors.initials: JD
authors.lastname: Wadsworth
authors.forename: C C
authors.initials: CC
authors.lastname: Shone
authors.forename: S
authors.initials: S
authors.lastname: Mochida
authors.forename: S
authors.initials: S
authors.lastname: Lande
authors.forename: J
authors.initials: J
authors.lastname: Melling
authors.forename: J O
authors.initials: JO
authors.lastname: Dolly
authors.forename: L
authors.initials: L
authors.lastname: Tauc
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D001048:Aplysia; D001905:Botulinum Toxins / Q000378:metabolism / Q000494:pharmacology*; D003964:Diaphragm / Q000294:innervation; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D009045:Motor Endplate / Q000187:drug effects / Q000502:physiology; D009046:Motor Neurons / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010791:Phrenic Nerve / Q000187:drug effects / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects; D013696:Temperature
chemical_list: D046911:Macromolecular Substances; D009498:Neurotoxins; D018377:Neurotransmitter Agents; D010446:Peptide Fragments; D000110:Acetylcholinesterase; D001905:Botulinum Toxins; D000109:Acetylcholine