Development of a Rapid Throughput Assay for Identification of hNa _v1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin

Abstract

Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Na _v1.1–Na _v1.9), Na _v1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Na _v1.7 becomes a promising target for analgesic drug development. In this study, we compared the influence of an array of VGSC agonists including veratridine, BmK NT1, brevetoxin-2, deltamethrin and antillatoxin (ATX) on membrane depolarization which was detected by Fluorescence Imaging Plate Reader (FLIPR) membrane potential (FMP) blue dye. In HEK-293 cells heterologously expressing hNa _v1.7 α-subunit, ATX produced a robust membrane depolarization with an EC ₅₀ value of 7.8 ± 2.9 nM whereas veratridine, BmK NT1, and deltamethrin produced marginal response. Brevetoxin-2 was without effect on membrane potential change. The ATX response was completely inhibited by tetrodotoxin suggesting that the ATX response was solely derived from hNa _v1.7 activation, which was consistent with the results where ATX produced a negligible response in null HEK-293 cells. Six VGSC antagonists including lidocaine, lamotrigine, phenytoin, carbamazepine, riluzole, and 2-amino-6-trifluoromethylthiobenzothiazole all concentration-dependently inhibited ATX response with IC ₅₀ values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNa _v1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNa _v1.7 antagonists.