[³H]-lifarizine, a high affinity probe for inactivated sodium channels

1 [³H]-lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (K(d) = 10.7 ± 2.9 nM; B(max) = 5.10 ± 1.43 pmol mg^-1 protein). 2 The binding of [³H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (a-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 µM had no effect on [³H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [³H]- batrachotoxinin-A 20-a-benzoate (BTX) binding with moderate affinity (pIC₅₀ 7.31 ± 0.24) indicating an interaction with toxin site 2. However, lifarizine accelerated the dissociation of [³H]-BTX and decreased both the affinity and density of sites labelled by [³H]-BTX, suggesting an allosteric interaction with toxin site 2. 3 The binding of [³H]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC₅₀ for cold lifarizine=7.99 ± 0.09 in membranes and 6.68 ± 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl increased the affinity of lifarizine almost 10 fold (pIC₅₀=7.86 ± 0.25). This suggests that lifarizine binds selectively to inactivated sodium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4 There was negligible [³H]-lifarizine and [³H]-BTX binding to solubilized sodium channels, although [³H]-STX binding was retained under these conditions. 5 The potencies of a series of compounds in displacing [³H]-lifarizine from rat cerebrocortical membranes correlated well with their affinities for inactivated sodium channels estimated from whole-cell voltage clamp studies in the mouse neuroblastoma cell line, N1E-115 (r=0.96). 6 These results show that [³H]-lifarizine is a high affinity ligand for neuronal sodium channels which potently and selectively labels a site, allosterically linked to toxin binding site 2, associated with inactivated sodium channels.