Ac. Mackinnon et al., [H-3] LIFARIZINE, A HIGH-AFFINITY PROBE FOR INACTIVATED SODIUM-CHANNELS, British Journal of Pharmacology, 115(6), 1995, pp. 1103-1109
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.