SPECIFIC BINDING OF THE NOVEL NA-SUBUNIT OF RAT-BRAIN NA+ CHANNELS( CHANNEL BLOCKER PD85,639 TO THE ALPHA)

The local anesthetic-like Na+ channel-blocking drug [H-3]PD85639 [alph a-([4-H-3]phenyl)-N-[3-(2,6-dimethyl-1 piperizinyl)-alpha-propyl][4-H- 3]benzeneacetamide] binds specifically to receptor sites on Na+ channe ls in intact synaptosomes and synaptosomal membranes, purified and rec onstituted Na+ channels, and type IIA Na+ channel alpha subunits expre ssed in the transfected Chinese hamster ovary cell line CNaIIA-1 No sp ecific binding was observed in nontransfected CHO-K1 cells, confirming the specificity of binding to Na+ channels. Two classes of binding si tes that differed in affinity and dissociation rate were observed in a ll three preparations. In synaptosomes, the high affinity sites had K( d) values of 3-20 nm and a B(max) of approximately 0.2 pmol/mg, wherea s the low affinity sites had K(d) values of 0.4-20 muM and a B(max) of approximately 5 pmol/mg. Binding of PD85,639 was inhibited by the loc al anesthetics tetracaine, bupivacaine, and mepivacaine at concentrati ons in the same range as those that inhibit Na+ channels. Tetracaine d id not affect the dissociation rate of PD85,639, consistent with compe titive binding of these two drugs at the same receptor site. In contra st, binding of PD85,639 was unaffected by the anticonvulsants phenytoi n and carbamazepine, which also inhibit Na+ channels. Veratridine and batrachotoxin, which bind at neurotoxin receptor site 2 on Na+ channel s, inhibited specific PD85,639 binding completely. PD85,639 accelerate d dissociation of specifically bound batrachotoxin, consistent with an indirect allosteric interaction between these two compounds. Thus, li ke local anesthetics, PD85,639 inhibits binding of batrachotoxin by an allosteric mechanism. The results indicate that PD85,639 binds specif ically to a local anesthetic receptor site on the Na+ channel alpha su bunit that is allosterically linked to neurotoxin receptor site 2. PD8 5,639 may be a useful molecular probe of this important drug receptor site on the Na+ channel.