Ca(sup 2+)-Dependent Noradrenaline Release from Permeabilised PC12 Cells IsBlocked by Botulinum Neurotoxin A or Its Light Chain

摘要

Permeabilisation of PC12 cells with digitonin allowed a direct study of theintracellular action of botulinum neurotoxin A, one of a group of dichain proteins produced by Clostridium botulinum that causes the fatal neuroparalytic condition, botulism. Release of 3Hnoradrenaline from these permeabilised cells could be evoked by Ca(sup 2+) and this was inhibited specifically by the neurotoxin in a dose-dependent manner (half-maximal dose 2nM under the conditions used). Inclusion of the reducing agent dithiothreitol (up to 10 mM) had no effect on the level of inhibition. Moreover, electrophoretic analysis showed that this treatment of the toxin in the native state caused negligible reduction of inter-chain disulphide bonds. Toxin-induced blockade of neurotransmitter releases was incomplete and could not be overcome by increased Ca2+ concentration (100 uM). The observed toxin-insensitivity of the release from intact PC12 cells must result from inefficient toxin uptake, relative to that in peripheral cholinergic neurones. Refolded light chain alone inhibited exocytosis to the same degree and with similar potency to that of the intact neurotoxin, and effect was not altered by the heavy chain.