High Affinity Binding of Pyrethroids to the a Subunit of Brain Sodium Channels

Na channels are the primary molecular targets of the pyrethroid insecticides. Na channels consisting of only a type IIA a 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 5 2139 mV) than unmodified Na channels (V0.5 5 228 mV). These results indicate that pyrethroids can modify the functional properties of the Na channel a 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 a subunit of voltage-gated Na channels, a radioactive photosensitive derivative, [H]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 KD values of ;58–300 nM for three representative pyrethroids. Specific pyrethroid binding was enhanced by ;40% in the presence of 100 nM a-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 [H]RU58487 under these optimal binding conditions revealed a radiolabeled band with an apparent molecular mass of 240 kDa corresponding to the Na channel a subunit. Anti-peptide antibodies recognizing sequences within the a 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 a subunit of Na channels and can bind specifically to a receptor site on the a subunit.