Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels
Label-Free Whole Cell Biosensing for High-Throughput Discovery of Activators and Inhibitors Targeting G Protein-Activated Inwardly Rectifying Potassium Channels
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DOI：
10.1021/acsomega.8b02254
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发表时间：
2018-11-01
期刊：
影响因子：
4.1
通讯作者：
Kostenis, Evi
中科院分区：
文献类型：
--
作者：
Krebs, Katrin M.;Pfeil, Eva M.;Kostenis, Evi
动态质量重分布(DMR)和细胞介电光谱(CDS)是一种无标记生物传感器技术，可捕获暴露在细胞外和细胞内刺激下的实时综合细胞反应。它们记录的信号通路伴随着细胞形状的改变和/或它们所连接的生物传感器近端细胞的分子运动。在这里，我们报告了一个意想不到的观察结果，即直接刺激G蛋白激活的内向整流钾(GIRK)通道也会引发强大的DMR和CDS信号，GIRK通道参与心脏和大脑中兴奋性的调节。使用小分子GIRK激活剂ML297、通道阻滞剂和细胞骨架网络抑制剂，我们发现GIRK激活通过一种依赖于肌动蛋白而不是微管网络的机制对细胞形态产生影响。由于无标记实时生物传感(I)定量确定GIRK激活剂的浓度依赖性，(Ii)准确评估GIRK通道阻滞剂的影响，(Iii)高通量兼容，(Iv)可视化先前未知的GIRK直接激活下游的细胞后果，我们不仅为GIRK配体的鉴定提供了一种新的实验策略，而且为探索GIRK(配体)生物学提供了一个全新的角度。我们预计，DMR和CDS可能会增加系统开发离子通道功能的技术，进而识别新的GIRK配体，以治疗心血管和神经疾病。
Dynamic mass redistribution (DMR) and cellular dielectric spectroscopy (CDS) are label-free biosensor technologies that capture real-time integrated cellular responses upon exposure to extra- and intracellular stimuli. They register signaling routes that are accompanied by cell shape changes and/or molecular movement of cells proximal to the biosensor to which they are attached. Here, we report the unexpected observation that robust DMR and CDS signatures are also elicited upon direct stimulation of G protein-activated inwardly rectifying potassium (GIRK) channels, which are involved in the regulation of excitability in the heart and brain. Using ML297, a small-molecule GIRK activator, along with channel blockers and cytoskeletal network inhibitors, we found that GIRK activation exerts its effects on cell shape by a mechanism which depends on actin but not the microtubule network. Because label-free real-time biosensing (i) quantitatively determines concentration dependency of GIRK activators, (ii) accurately assesses the impact of GIRK channel blockers, (iii) is high throughput-compatible, and (iv) visualizes previously unknown cellular consequences downstream of direct GIRK activation, we do not only provide a novel experimental strategy for identification of GIRK ligands but also an entirely new angle to probe GIRK (ligand) biology. We envision that DMR and CDS may add to the repertoire of technologies for systematic exploitation of ion channel function and, in turn, to the identification of novel GIRK ligands in order to treat cardiovascular and neurological disorders.