兴奋性氨基酸转运体(EAAT/SLC1)介导Na+依赖性胞外谷氨酸摄取,是神经系统疾病的潜在药物靶标。评估体外谷氨酸转运的常规方法是基于放射性标记,荧光染料或电生理学,这可能会损害细胞的生理机能,并且通常不太适合用于主要药物筛选。这里,我们描述了一种新的无标记方法来评估活细胞中的人类EAAT功能,即,不使用对底物或细胞环境的化学修饰。在过表达EAAT1的贴壁HEK293细胞中,用谷氨酸或天冬氨酸刺激诱导的细胞扩散,使用基于阻抗的生物传感器实时检测。在Na+/K+-ATP酶抑制剂乌巴因和EAAT抑制剂的存在下,细胞形态的这种变化被阻止。这表明底物诱导的反应是离子依赖性和转运蛋白特异性的。肌动蛋白细胞骨架重塑和渗透压牛磺酸细胞内水平的变化证实了表型反应的机理解释,这表明这种反应涉及细胞肿胀。此外,对于表达其他EAAT亚型的细胞,观察到底物诱导的细胞反应,以及在具有内源性EAAT1表达的乳腺癌细胞系(MDA-MB-468)中。这些发现允许开发无标记的高通量筛选试验,这可能有利于EAAT的早期药物发现,并具有研究调节细胞形状的其他转运蛋白的潜力。
Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell\'s physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape.