Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin.

Fang Zhao,Zhengyu Cao,Liang Jin,Boyang Yu,Fan Zhang,Xichun Li,Masayuki Inoue

doi:10.3390/md14020036

Fang Zhao, Zhengyu Cao + Show 5 more

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https://doi.org/10.3390/md14020036

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Abstract

Voltage-gated sodium channels (VGSCs) are responsible for the generation of the action potential. Among nine classified VGSC subtypes (Nav1.1–Nav1.9), Nav1.7 is primarily expressed in the sensory neurons, contributing to the nociception transmission. Therefore Nav1.7 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 hNav1.7 α-subunit, ATX produced a robust membrane depolarization with an EC50 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 hNav1.7 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 IC50 values comparable to that reported from patch-clamp experiments. Considered together, we demonstrate that ATX is a unique efficacious hNav1.7 activator which offers a useful probe to develop a rapid throughput screening assay to identify hNav1.7 antagonists.

Highlights

Voltage-gated sodium channels (VGSCs) are responsible for the rising phase of the action potential in excitable cells such as neurons, cardiac myocytes and skeletal muscle myocytes [1,2].VGSCs are composed of voltage-sensing and pore-forming elements in one principal α-subunit and one or two auxiliary β-subunits which alter the channel physiological properties and subcellularMar
We further demonstrated that deltamethin, which bound to an undefined neurotoxin site delaying the inactivation of the VGSCs [32], produced minimal response on the membrane potential changes
We demonstrated that the IC50 values for lamotrigine, carbamazepine and phenytoin suppression of ATX-induced membrane depolarization in hNav 1.7-Human Embryonic Kidney 293 (HEK-293) cells were 66.3, 77.7 and 18.7 μM, respectively, which were more consistent with their affinities on the inactivated state

Summary

Introduction

Voltage-gated sodium channels (VGSCs) are responsible for the rising phase of the action potential in excitable cells such as neurons, cardiac myocytes and skeletal muscle myocytes [1,2].VGSCs are composed of voltage-sensing and pore-forming elements in one principal α-subunit and one or two auxiliary β-subunits which alter the channel physiological properties and subcellularMar. VGSCs represent the molecular targets for a broad range of potent neurotoxins that bind to at least six distinct neurotoxin sites on the sodium channel α-subunit and affect the ion permeation and gating of sodium channels [3]. These toxins include tetrodotoxin (TTX), saxitoxin, and μ-conotoxin (site 1); lipid-soluble alkaloid toxins, including batrachotoxin, veratridine, aconitine, and grayanotoxin (site 2); polypeptide sea anemone and α-scorpion toxins (site 3); β-scorpion toxins (site 4); marine toxins such as brevetoxins (PbTxs) and ciguatoxins (site 5); and δ-conotoxins (site 6) [5]. Pyrethroid insecticides act at a distinctive site on the sodium channel α-subunit to enhance channel activity by shifting activation to more negative membrane potentials as well as by delaying inactivation [6]

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