Abstract

Na⁺ channels are the primary molecular targets of the pyrethroid insecticides. Na⁺ channels consisting of only a type IIA α 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 (V _0.5 = −139 mV) than unmodified Na⁺ channels (V _0.5 = −28 mV). These results indicate that pyrethroids can modify the functional properties of the Na⁺ channel α 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 α 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°, dissociated with a half-time of ∼10 min, and had K _D values of ∼58–300 nm for three representative pyrethroids. Specific pyrethroid binding was enhanced by ∼40% in the presence of 100 nm α-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 α subunit. Anti-peptide antibodies recognizing sequences within the α 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 α subunit of Na⁺ channels and can bind specifically to a receptor site on the α subunit.