High affinity binding of pyrethroids to the alpha subunit of brain sodium channels.

Na+ channels are the primary molecular targets of the pyrethroid insecticides. Na+ channels consisting of only a type IIA alpha subunit expressed 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+ current decayed slowly on repolarization. These modified Na+ channels could be reactivated at much more negative membrane potentials (V0.5 = -139 mV) than unmodified Na+ channels (V0.5 = -28 mV). These results indicate that pyrethroids can modify the functional properties of the Na+ channel alpha subunit expressed alone by blocking their inactivation, shifting their voltage dependence of activation, and slowing their deactivation. To demonstrate directly the specific interaction of pyrethroids with the alpha subunit of voltage-gated Na+ channels, a radioactive photosensitive derivative, [3H]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+ channels in rat brain membrane preparations could be measured and reached 75% of total binding under optimal conditions. Binding approached equilibrium within 1 hr at 4 degrees, dissociated with a half-time of approximately 10 min, and had K(D) values of approximately 58-300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced by approximately 40% in the presence of 100 nM alpha-scorpion toxin, but no allosteric enhancement was observed in the presence of toxins acting at other Na+ channel receptor sites. Extensive membrane washing increased specific binding to 89%. Photolabeling with [3H]RU58487 under these optimal binding conditions revealed a radiolabeled band with an apparent molecular mass of 240 kDa corresponding to the Na+ channel alpha subunit. Anti-peptide antibodies recognizing sequences within the alpha subunit were able to specifically immunoprecipitate the covalently modified channel. Together, these results demonstrate that the pyrethroids can modify the properties of cells expressing only the alpha subunit of Na+ channels and can bind specifically to a receptor site on the alpha subunit.