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

Abstract

A new method is used to tritiate tetrodotoxin:starting with tetrodotoxin, acetylanhydrotetrodotoxin is formed which is then reacted in T 2O 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.