Specific binding of the novel Na+ channel blocker PD85,639 to the alpha subunit of rat brain Na+ channels.

The local anesthetic-like Na+ channel-blocking drug [3H]PD85639 [alpha-([4-3H]phenyl)-N-[3-(2,6-dimethyl-1-piperizinyl)-alpha-prop yl] [4-3H]benzeneacetamide] binds specifically to receptor sites on Na+ channels in intact synaptosomes and synaptosomal membranes, purified and reconstituted Na+ channels, and type IIA Na+ channel alpha subunits expressed in the transfected Chinese hamster ovary cell line CNaIIA-1. No specific binding was observed in nontransfected CHO-K1 cells, confirming the specificity of binding to Na+ channels. Two classes of binding sites that differed in affinity and dissociation rate were observed in all three preparations. In synaptosomes, the high affinity sites had Kd values of 3-20 nM and a Bmax of approximately 0.2 pmol/mg, whereas the low affinity sites had Kd values of 0.4-20 microM and a Bmax of approximately 5 pmol/mg. Binding of PD85,639 was inhibited by the local anesthetics tetracaine, bupivacaine, and mepivacaine at concentrations in the same range as those that inhibit Na+ channels. Tetracaine did not affect the dissociation rate of PD85,639, consistent with competitive binding of these two drugs at the same receptor site. In contrast, binding of PD85,639 was unaffected by the anticonvulsants phenytoin and carbamazepine, which also inhibit Na+ channels. Veratridine and batrachotoxin, which bind at neurotoxin receptor site 2 on Na+ channels, inhibited specific PD85,639 binding completely. PD85,639 accelerated dissociation of specifically bound batrachotoxin, consistent with an indirect allosteric interaction between these two compounds. Thus, like local anesthetics, PD85,639 inhibits binding of batrachotoxin by an allosteric mechanism. The results indicate that PD85,639 binds specifically to a local anesthetic receptor site on the Na+ channel alpha subunit that is allosterically linked to neurotoxin receptor site 2. PD85,639 may be a useful molecular probe of this important drug receptor site on the Na+ channel.