Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin.
Development of a Rapid Throughput Assay for Identification of hNav1.7 Antagonist Using Unique Efficacious Sodium Channel Agonist, Antillatoxin.
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使用独特的有效钠通道激动剂抗鱼毒素开发快速通量测定法来鉴定 hNa(v)1.7 拮抗剂
DOI：
10.3390/md14020036
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发表时间：
2016-02-16
期刊：
影响因子：
5.4
通讯作者：
Cao Z
中科院分区：
文献类型：
--
作者：
Zhao F;Li X;Jin L;Zhang F;Inoue M;Yu B;Cao Z
电压门控钠通道（VGSCs）负责动作电位的产生。在9种VGSC亚型（Nav1.1-Nav1.9）中，Nav1.7主要表达于感觉神经元，参与伤害感觉的传递。因此，Nav1.7成为镇痛药物开发的重要靶点。在这项研究中，我们比较了一系列VGSC激动剂包括veratridine， BmK NT1, brevetoxin-2， deltamethrin和antillatoxin （ATX）对荧光成像板阅读器（FLIPR）膜电位（FMP）蓝染料检测膜去极化的影响。在异种表达hNav1.7 α-亚基的HEK-293细胞中，ATX具有较强的膜去极化效应，EC50值为7.8±2.9 nM，而veratridine、BmK NT1和溴氰菊酯的膜去极化效应较弱。Brevetoxin-2对膜电位变化无影响。河豚毒素完全抑制了ATX反应，这表明ATX反应完全来自于hNav1.7的激活，这与ATX在空HEK-293细胞中产生可忽略不计的反应的结果一致。6种VGSC拮抗剂，包括利多卡因、拉莫三嗪、苯妥英、卡马西平、利鲁唑和2-氨基-6-三氟甲基硫代苯并噻唑，均呈浓度依赖性地抑制ATX反应，IC50值与膜片钳实验的IC50值相当。综上所述，我们证明了ATX是一种独特有效的hNav1.7激活剂，它为开发一种快速通量筛选试验来鉴定hNav1.7拮抗剂提供了有用的探针。
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.