Lummis Sarah C R, Salvage Samantha C, Huang Christopher L-H, Jackson Antony P
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK.
Physiology, Development and Neuroscience, University of Cambridge, Downing Place, Cambridge CB2 3DY, UK.
Membranes (Basel). 2025 Mar 5;15(3):80. doi: 10.3390/membranes15030080.
Voltage-gated sodium channels (Navs) are critical for membrane potential depolarisation in cells, with especially important roles in neuronal and cardiomyocyte membranes. Their malfunction results in a range of disorders, and they are the target of many widely used drugs. A rapid yet accurate functional assay is therefore desirable both to probe for novel active compounds and to better understand the many different Nav isoforms. Here, we use fluorescence to monitor Nav function: cells expressing either the cardiac Nav 1.5 or pain-associated Nav 1.7 were loaded with fluorescent membrane potential sensitive dye and then stimulated with veratridine. Cells expressing Nav 1.5 show a concentration-dependent slow rise and then a plateau in fluorescence. In contrast, cells expressing Nav 1.7 show a more rapid rise and then unexpected oscillatory behavior. Inhibition by flecainide and mexiletine demonstrates that these oscillations are Nav-dependent. Thus, we show that this fluorescent membrane potential dye can provide useful functional data and that we can readily distinguish between these two Nav isoforms because of the behavior of cells expressing them when activated by veratridine. We consider these distinct behaviors may be due to different interactions of veratridine with the different Nav isoforms, although more studies are needed to understand the mechanism underlying the oscillations.
电压门控钠通道（Navs）对于细胞的膜电位去极化至关重要，在神经元和心肌细胞膜中发挥着尤为重要的作用。其功能异常会导致一系列疾病，并且它们是许多广泛使用药物的作用靶点。因此，无论是用于探寻新型活性化合物还是更好地理解多种不同的Nav亚型，都需要一种快速且准确的功能检测方法。在此，我们利用荧光来监测Nav的功能：将表达心脏型Nav 1.5或与疼痛相关的Nav 1.7的细胞加载荧光膜电位敏感染料，然后用藜芦碱进行刺激。表达Nav 1.5的细胞呈现出浓度依赖性的荧光缓慢上升，随后达到平稳状态。相比之下，表达Nav 1.7的细胞荧光上升更为迅速，随后出现意外的振荡行为。氟卡尼和美西律的抑制作用表明这些振荡是Nav依赖性的。因此，我们表明这种荧光膜电位染料能够提供有用的功能数据，并且由于表达它们的细胞在被藜芦碱激活时的行为，我们能够轻易区分这两种Nav亚型。我们认为这些不同的行为可能是由于藜芦碱与不同Nav亚型的相互作用不同所致，尽管还需要更多研究来了解振荡背后的机制。
