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

Abstract

[3H]‐lifarizine bound saturably and reversibly to an apparently homogeneous class of high affinity sites in rat cerebrocortical membranes (Kd= 10.7 ± 2.9 nM; Bmax = 5.10±1.43 pmol mg−1 protein). The binding of [3H]‐lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (α‐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‐α‐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. 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 KC1 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. There was negligible [3H]‐lifarizine and [3H]‐BTX binding to solubilized sodium channels, although [3H]‐STX binding was retained under these conditions. 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). 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. 1995 British Pharmacological Society