Radioactive Labelling of Toxin I fromAnemonia sulcataand Binding to Crayfish Nerve in vitro

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Abstract
Radioactive derivatives of neurotoxin I (ATX I) from A. sulcata were synthesized. Iodination of ATX I with 125I yielded a mixture of reaction products from which monoiodo and diiodo ATX I were isolated. 125I-ATX I bound to the axonal membrane from Astacus leptodactylus main walking nerve. Specificity of binding was shown by saturability of the binding sites and by competitive binding of native and radioactive toxin. Astacus nerve bound 44 fmol of 125I-ATX I/mg nerve (wet wt). The axonal membrane surface of the nerve was 7800 cm2/g nerve. This amounts to a binding site density of around 35/.mu.2 axonal surface. Binding was not inhibited by tetrodotoxin, the blocker of the selectivity filter of voltage-dependent Na channels. 125I-ATX I may bind to the Na channel-inactivating gate. The affinity of the nerve membrane receptors for 125I-ATX I appears to be voltage-dependent: Kd = 5 nM was found with whole crayfish nerves in the presence of tetrodotoxin, Kd = 40nM in the absence of tetrodotoxin and an even lower affinity was obtained with axonal membrane fragments isolated from the nerve. Drugs destabilizing the membrane potential, e.g., veratridine, ouabain and sodium azide, lowered the affinity or abolished binding completely.