[H-3] LIFARIZINE, A HIGH-AFFINITY PROBE FOR INACTIVATED SODIUM-CHANNELS

1 [H-3]-lifarizine bound saturably and reversibly to an apparently hom ogeneous 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 [H-3]-lifarizine was unaffected by sodium channel toxi ns binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 1 0 mu M had no effect on [H-3]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [H-3]-batrachotoxinin-A 20-alpha-benzoate (BTX) binding with moderate affinity (pIC(50) 7.31 +/- 0.24) indicating an i nteraction with toxin site 2. However, lifarizine accelerated the diss ociation of [H-3]-BTX and decreased both the affinity and density of s ites labelled by [H-3]-BTX, suggesting an allosteric interaction with toxin site 2. 3 The binding of [H-3]-lifarizine was voltage-sensitive, binding to membranes with higher affinity than to synaptosomes (pIC(5 0) for cold lifarizine = 7.99 +/- 0.09 in membranes and 6.68 +/- 0.14 in synaptosomes). Depolarization of synaptosomes with 130 mM KCl incre ased the affinity of lifarizine almost 10 fold (pIC(50) = 7.86 +/- 0.2 5). This suggests that lifarizine binds selectively to inactivated sod ium channels which predominate both in the membrane preparation and in the depolarized synaptosomal preparation. 4 There was negligible [H-3 ]-lifarizine and [H-3]-BTX binding to solubilized sodium channels, alt hough [H-3]-STX binding was retained under these conditions. 5 The pot encies of a series of compounds in displacing [H-3]-lifarizine from ra t cerebrocortical membranes correlated well with their affinities for inactivated sodium channels estimated from whole-cell voltage clamp st udies in the mouse neuroblastoma cell line, N1E-115 (r = 0.96). 6 Thes e results show that [H-3]-lifarizine is a high affinity ligand for neu ronal sodium channels which potently and selectively labels a site, al losterically linked to toxin binding site 2, associated with inactivat ed sodium channels.