[H-3] PHENYTOIN IDENTIFIES A NOVEL ANTICONVULSANT-BINDING DOMAIN ON VOLTAGE-DEPENDENT SODIUM-CHANNELS

The voltage-dependent sodium channel has been proposed as a specific target for the actions of the anticonvulsant drug phenytoin. Working at 0-4-degrees, we previously reported the existence of specific [H-3]phenytoin binding sites in rat brain membranes. In the present study, the binding of [H-3]phenytoin was assessed at 22-degrees, a temperature favorable to the binding of sodium channel ligands. At 22-degrees, the site had a K(d) of 1.5 muM, which is in the relevant therapeutic concentration range for anticonvulsant activity (1-10 muM), and a calculated B(max) of 4.5 pmol/mg of protein, which is similar to previous estimates of sodium channel concentration in brain membranes. In competition experiments, specific [H-3]phenytoin binding was found to be inhibited by drugs that interact with the sodium channel, including antiarrhythmics, local anesthetics, anticonvulsants, and site-specific neurotoxins (the steroidal alkaloid activators, beta-scorpion venoms, and brevetoxin-3). Diazepam, used clinically in the management of tonic-clonic status epilepticus, and flunarizine, a calcium channel blocker with anticonvulsant activity, potentiated [H-3]phenytoin binding at micromolar concentrations. Other drugs and ligands, including neurotransmitters, neuromodulators, and ligands for other ion channels, had no effect. Depolarization with KCl showed [H-3]phenytoin binding to be voltage sensitive. Experiments with batrachotoxin (a specific site 2 toxin) and anticonvulsants demonstrated that the interactions between these compounds and the [H-3]phenytoin binding site are allosteric in nature. These results provide direct evidence that phenytoin interacts with the voltage-dependent sodium channel and indicate that such interactions take place at therapeutic concentrations. They support previous proposals, based on toxin-binding and electrophysiological studies, that the therapeutic effects of phenytoin result from a selective inhibition of voltage-dependent sodium flux.