Abstract

The effectiveness of several antiepileptic, analgesic, and neuroprotective drugs is attributable to state-dependent inhibition of voltage-gated sodium channels. To help characterize their site and mode of action on sodium channels, a member of the lamotrigine family, R-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)-pyrimidine (BW202W92), was radiolabeled and used as a binding ligand in rat forebrain synaptosomes. Although the level of specific [³H]BW202W92 binding in a standard incubation medium was relatively poor, low concentrations of tetrodotoxin (EC₅₀ = 2-3 nM) greatly enhanced the binding, apparently by increasing the affinity of the binding sites. Tetrodotoxin-dependent binding was stereoselective (the less active enantiomer, S-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)-pyrimidine (BW203W92), was up to 30-fold less potent, depending on conditions) and was extremely sensitive to inhibition by raised K⁺ concentration (IC₅₀ = 5.9 mM), an effect that was ascribed to changes in membrane potential. In addition, the binding was inhibited by sodium channel neurotoxins acting on sites 3 and 4, but it was resistant to batrachotoxin (site 2) and brevetoxin (site 5). Several drugs acting on sodium channels displaced tetrodotoxin-dependent [³H]BW202W92 binding, and most of those tested showed different affinities under depolarized (100 mM K⁺) and polarized (1 mM K⁺) conditions. In a subset of compounds for which data were available, binding affinity in depolarized synaptosomes correlated well with apparent affinity for the inactivated state of sodium channels. The [³H]BW202W92 binding site is novel and is likely to represent a pharmacologically important site of action of drugs on voltage-gated sodium channels in the brain.