HIGH-AFFINITY BINDING OF PYRETHROIDS TO THE ALPHA-SUBUNIT OF BRAIN SODIUM-CHANNELS

Na+ channels are the primary molecular targets of the pyrethroid insec ticides. Na+ channels consisting of only a type IIA alpha subunit expr essed in Chinese hamster ovary cells responded to pyrethroid treatment in a normal manner: a sustained Na+ current was induced progressively after each depolarizing pulse in a train of stimuli, and this Na+ cur rent decayed slowly on repolarization. These modified Na+ channels cou ld be reactivated at much more negative membrane potentials (V-0.5 = - 139 mV) than unmodified Na+ channels (V-0.5 = -28 mV). These results i ndicate that pyrethroids can modify the functional properties of the N a+ channel alpha subunit expressed alone by blocking their inactivatio n, shifting their voltage dependence of activation, and slowing their deactivation. To demonstrate directly the specific interaction of pyre throids with the alpha subunit of voltage-gated Na+ channels, a radioa ctive photosensitive derivative, [H-3]RU58487, was used in binding and photolabeling studies. In the presence of a low concentration of the nonionic detergent Triton X-100, specific pyrethroid binding to Na+ ch annels in rat brain membrane preparations could be measured and reache d 75% of total binding under optimal conditions. Binding approached eq uilibrium within 1 hr at 4 degrees, dissociated with a half-time of si milar to 10 min, and had K-D values of similar to 58-300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced b y similar to 40% in the presence of 100 nM alpha-scorpion toxin, but n o allosteric enhancement was observed in the presence of toxins acting at other Na+ channel receptor sites. Extensive membrane washing incre ased specific binding to 89%. Photolabeling with [H-3]RU58487 under th ese optimal binding conditions revealed a radiolabeled band with an ap parent molecular mass of 240 kDa corresponding to the Na+ channel a su bunit. Anti-peptide antibodies recognizing sequences within the a subu nit were able to specifically immunoprecipitate the covalently modifie d channel. Together, these results demonstrate that the pyrethroids ca n modify the properties of cells expressing only the a subunit of Nachannels and can bind specifically to a receptor site on the alpha sub unit.