Chemically tritiated tetrodotoxin: physiological activity and binding to Na-channels

A new method is used to tritiate tetrodotoxin:starting with tetrodotoxin, acetylanhydrotetrodotoxin is formed which is then reacted in $\text{T}{}_2\text{O}\text{TCl}$ to ³H]tetrodotoxin. The formation of the intermediate and of the tritiated product is analytically monitored by bioassay. After purification ³H]tetrodotoxin is obtained at a specific activity of 18 Ci/mmol. No back exchange of tritium was observed under physiological conditions. The binding of ³H]tetrodotoxin to voltage-sensitive Na channels was studied with membrane fragments from Electrophorus electricus electric organ. Binding studies were carried out by variation of the concentration of ³H]tetrodotoxin and by competition between ³H]tetrodotoxin and reference tetrodotoxin. The apparent dissociation constant for binding to Na channels in these membrane fragments is K_D = (20 ± 10) nM. In contrast, ³H]tetrodotoxin blocks Na current in Rana esculenta nodes with an apparent K_D = 3 nM. The difference may be due to a higher density of negative surface charges at the nodal regions.