Insect nervous system [3H]saxitoxin binding sites: characterization and target size

Abstract

Saxitoxin (STX) has proved useful in the isolation and characterization of vertebrate and invertebrate voltage-operated sodium channels. Membrane extracts from the nervous system of the cockroach Periplaneta americana contain a saturable component of specific [(3)H]STX binding. Scatchard analysis yields a K(D) of 0.84 nM, similar to that (3.0 nM) determined in electrophysiological studies on axons in the same tissue (Sattelle et al., 1979). The maximum number of binding sites, B(max) (8.25 pmol/mg protein), was higher than previously observed. The specific binding component was blocked by STX and tetrodotoxin (TTX), but not by scorpion (Leiurus quinquestriatus) venom, aconitine, veratridine, sea anemone toxin and deltamethrin, which act at different sites on the channel molecule. Unlabelled STX samples prepared from different sources (Mytilus, Saxidomus and Gonyaulax) were all effective as inhibitors of [(3)H]STX binding. Radiation inactivation was employed to determine the molecular target size of the [(3)H]STX binding molecule in membranes prepared from the cockroach nervous system. By this means M(r) = 171,400 +/- 25,000 was estimated for the insect sodium channel.