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

Abstract

1. [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). 2. The binding of [3H]-lifarizine was unaffected by sodium channel toxins binding to site 1 (tetrodotoxin), site 3 (alpha-scorpion venom) or site 5 (brevetoxin), Furthermore, lifarizine at concentrations up to 10 microM had no effect on [3H]-saxitoxin (STX) binding to toxin site 1. Lifarizine displaced [3H]-batrachotoxinin-A 20-alpha-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, NIE-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 within activated sodium channels.

• Authors: A C MacKinnon, K M Wyatt, J G McGivern, R D Sheridan, C M Brown
• Journal: British journal of pharmacology (Br J Pharmacol) Vol. 115 Issue 6 Pg. 1103-9 (Jul 1995) ISSN: 0007-1188 [Print] England
• PMID: 7582509 (Publication Type: Journal Article)

Chemical References

• Imidazoles
• Piperazines
• Sodium Channels
• Saxitoxin
• lifarizine

Topics

• Animals
• Cerebral Cortex (drug effects, metabolism)
• Dose-Response Relationship, Drug
• Imidazoles (metabolism, pharmacology)
• Male
• Patch-Clamp Techniques
• Piperazines (metabolism, pharmacology)
• Radioligand Assay
• Rats
• Rats, Sprague-Dawley
• Saxitoxin (pharmacology)
• Sodium Channels (drug effects, metabolism)
• Time Factors