Brevetoxins, isolated from the marine dinoflagellate Ptychodiscus brevis, exert their effects by altering the membrane resting potential of excitable cell types in ways that activate sodium channels at normal resting potential. The interaction between the lipid-soluble polyether ladder brevetoxins and Site 5 associated with voltage- sensitive sodium channels will be explored using synthetic toxin fragments and chemically-modified natural toxins produced in cultures, biochemical radioligand and radioactive ion flux protocols in rat brain synaptosomes, and electrophysiological voltage-clamp measurements in isolated rat parasympathetic neurons. Using computer modeling, the conformation of the toxins and their derivatives will be evaluated, and a model of the structural features necessary for binding and activity will be developed and refined. Synthetic toxin surrogates will be synthesized and evaluated using biochemical and electrophysiological techniques.Using the binding, ion flux, and electrophysiological results for each derivative, in conjunction with computer modeling, interactive refinement of an hypothetical receptor for the toxin backbone will be carried out. The results of this work will be used together with toxin photoaffinity protocols, immunoprecipitation studies, biochemical purification of the brevetoxin-specific binding site, and primary sequence data to generate a more complete picture of how this new class of toxic ligands interacts with the alpha-subunit of the voltage-sensitive sodium channel. This collaborative proposal brings together a reliable source of natural brevetoxins, the synthetic expertise to modify their structures and to synthesize surrogates, the latest in molecular modeling techniques, and the abilities to measure toxin binding and sodium ion influx biochemically and ionic conductances electrophysiologically. The long-term goal is to gain insight into the mechanism of sodium ion channel gating, to produce readily available probes that will modulate sodium ion flux in predictable ways, and to gain insight into the development of potential brevetoxin and ciguatoxin antagonists for therapeutic intervention.
