CHELATION OF ZINC ANTAGONIZES THE NEUROMUSCULAR BLOCKING PROPERTIES OF THE 7 SEROTYPES OF BOTULINUM NEUROTOXIN AS WELL AS TETANUS TOXIN

Botulinum neurotoxin types A, B (unactivated and activated), C, D, E, F and G, as well as tetanus toxin, paralyzed transmission in mouse phr enic nerve-hemidiaphragm preparations. Toxin-induced blockade of trans mission was antagonized by chelators [e.g., ethylenediamine tetraaceti c acid, tetrakis(2-pyridylmethyl) ethylenediamine or diethylene-triami nepentaacetic anhydride], but this effect was dependent on incubation conditions. Pretreatment of toxin with chelators failed to produce ant agonism, but pretreatment of tissues did produce antagonism. Of the va rious chelators tested, tetrakis(2-pyridylmethyl)ethylenediamine produ ced the greatest effect. Antagonism of toxin-induced neuromuscular blo ckade could be partially reversed by washing chelators from tissues an d could be fully reversed by adding an excess of zinc. The ability of chelators to antagonize clostridial neurotoxins was specific and did n ot extend to phospholipase A2 neurotoxins. Ligand-binding studies with radioiodinated toxin and brain membrane preparations showed that chel ators did not antagonize toxicity by inhibiting toxin association with receptors. Similarly, pharmacological experiments with unlabeled toxi n- and type-specific antibodies demonstrated that chelators did not ac t by blocking receptor-mediated internalization of toxin. The chelator s appeared to exert their effects by antagonizing the intracellular ac tions of clostridial neurotoxins. Electrophysiological studies showed that chelators, at concentrations relevant to antagonism of botulinum neurotoxin and tetanus toxin, did not enhance transmitter release. The refore, the chelators appeared to work by inhibiting the proposed zinc endoprotease activity of clostridial neurotoxins. The results indicat e that there are three classes of universal antagonists that delay or abolish the actions of all serotypes of botulinum neurotoxin and tetan us toxin: 1) lectins with affinity for sialic acid antagonize binding, 2) drugs that block (e.g., bafilomycin) or reverse (e.g., methylamine hydrochloride) acidification of endosomes antagonize internalization and 3) drugs that chelate zinc antagonize intracellular expression of toxicity. The three classes of universal antagonists can be used in co mbination to produce striking reductions in the apparent potency of cl ostridial neuro toxins.