Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
Mater Research Institute-The University of Queensland, Faculty of medicine, The University of Queensland, Woolloongabba, QLD, Australia.
PLoS One. 2019 Mar 11;14(3):e0213751. doi: 10.1371/journal.pone.0213751. eCollection 2019.
Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 μM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.
电压门控钠离子通道（NaV）是疼痛、癫痫和心律失常的重要治疗靶点。在这里，我们描述了一种无需洗涤的荧光钠离子内流测定法的开发，该方法适用于高通量筛选和新型药物先导物的表征。在异源表达 hNaV1.1-1.8 的 HEK293 细胞中，向测定法中加入红色-紫色食品染料（峰值吸收范围 495-575nm），可有效猝灭钠离子指示剂染料 Asante NaTRIUM Green-2（ANG-2；峰值发射 540nm）的背景荧光，从而无需洗涤步骤。胭脂红 4R（1mM）被鉴定为合适的猝灭剂，如通过膜片钳实验评估的那样，它对 NaV 通道没有直接作用，并且不会改变 NaV1.1-1.7 激活剂藜芦碱（EC50 为 10-29μM）或 NaV1.1-1.8 抑制剂四卡因（IC50 为 6-66μM）的药理学。此外，我们还发现食品染料胭脂红 4R、亮黑 BN、诱惑红和苋菜红有效地猝灭钙指示剂染料 fluo-4、fura-2 和 fura-5F 的背景荧光，表明它们可能是无需洗涤的钙离子指示剂试剂盒的廉价替代品。总之，我们已经开发了一种无需洗涤的荧光钠离子内流测定法，该方法基于钠离子指示剂染料 ANG-2 和猝灭剂胭脂红 4R，适用于高通量筛选。
