J. Oliver Dolly

Characterization of the Inhibitory Action of Botulinum Neurotoxin Type A on the Release of Several Transmitters from Rat Cerebrocortical Synaptosomes

Abstract: Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit ∼90% of Ca2+‐dependent K+‐evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose‐response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at ∼10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+‐independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+‐dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H‐catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4‐aminopyridine could reverse the toxin‐induced blockade of evoked release. This lack of specificity in its action on synaptosomes. and other published findings, lead to the conclusion that toxin‐sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.

Identification by cross-linking of a neuronal acceptor protein for dendrotoxin, a convulsant polypeptide.

Dendrotoxin, a lijow molecular weight protein from the venom of Dendroaspis angusticeps, is known to be a potent convulsant that attenuates one type of voltage‐sensitive K+ channel in guinea‐pig hippocampus. A biologically active preparation of 125I‐labelled dendrotoxin has been cross‐linked to its high‐affinity protein acceptor in synaptic plasma membranes from rat cerebral cortex. On SDS gel electrophoresis, a complex with a Mr of 72,000 was observed which, assuming one toxin molecule is attached, yields an apparent size of 65,000 for this subunit of the acceptor. Unlike dendrotoxin, low concentrations of β‐bungarotoxin, another pre‐synaptically acitve toxin, do not inhibit its labelling.

Distribution of acceptors for ß-bungarotoxin in the central nervous system of the rat

High-affinity acceptors for 125I-beta-bungarotoxin have been identified on rat brain cryostat sections and mapped quantitatively using 3H-sensitive sheet film autoradiography. A unique distribution of acceptors has thus been observed; the toxin sites are particularly enriched in grey matter areas and synaptic regions, consistent with the pharmacological action of beta-bungarotoxin. As the binding was abolished by dendrotoxin, a related polypeptide known to inhibit fast-activating K+ conductances, the occurrence of beta-bungarotoxin acceptors may indicate the location of certain voltage-sensitive K+ channels. The overall distribution is, however, distinct from that of any other ion channel described.

Peripheral and central nicotinic ACh receptors — how similar are they?

Abstract Study of the nicotinic acetylcholine receptor (nAChR) from fish electric organs has now reached an exceptional degree of sophistication. For example, partial N-terminal amino acid sequences of the nAChRs four subunits and their arrangement in the membrane are now known 9,16 , the interactions between the nAChR and its agonists have been studied using rapid-reaction kinetics, and the receptor ion-channel has been reconstituted in artificial membranes (reviewed in Refs 6, 8, 10). For neurobiologists the nAChRs of vertebrate muscle and brain are clearly of greater interest. However, in these tissues nAChR concentration is 1000-fold or more lower and, due to the variety of proteases released on extraction 13,19 , the receptors stability is also low. Thus, progress in this field has been slower. Here, Eric Barnard and Oliver Dolly compare the receptor from these diverse sources. They note also the relevance of another recent advance: this receptor is the first to which mRNA and recombinant DNA techniques have been successfully applied.

Dendrotoxin and charybdotoxin increase the cytosolic concentration of free Ca2+ in cerebrocortical synaptosomes: An effect not shared by apamin

Nanomolar concentrations of charybdotoxin or dendrotoxin increase the cytoplasmic free Ca2+ concentration in isolated central nerve terminals. The effects of the two toxins, normally considered to be blockers of K+ channels controlled by voltage in a Ca2+ ‐sensitive or ‐insensitive manner, respectively, show only marginal additivity. Apamin, an inhibitor of low conductance Ca2+ ‐activated K+ channels, was without effect in either the absence or presence of dendrotoxin. The effect of charybdotoxin on polarized, isolated central nerve terminals seems to be mediated largely by a block of K+ channels sensitive to dendrotoxin. Apparently, these voltage‐operated K+ channels make a more significant contribution to maintaining the polarized potential of synaptosomes than do those activated by Ca2+.