Optical and Electronic Ion Channel Monitoring from Native Human Membranes
Optical and Electronic Ion Channel Monitoring from Native Human Membranes
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天然人膜的光电离子通道监测
DOI：
10.1021/acsnano.0c01330
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发表时间：
2020-10-27
期刊：
影响因子：
17.1
通讯作者：
Owens, Roisin M.
中科院分区：
文献类型：
--
作者：
Pappa, Anna-Maria;Liu, Han-Yuan;Owens, Roisin M.
跨膜蛋白是调节细胞活性的主要靶点，在治疗药物和病原体相互作用方面都是如此。筛选这种疗法或识别毒素的工作受到严重限制，因为缺乏可用的方法来提供关于功能的高含量信息(理想的多模式)和适合高通量的方法。在这里，我们已经展示了一个能够多模式(光学和电子)筛选人类来源的膜上的配基门控离子通道活性的平台。Trek-1离子通道在支持的脂质双层中表达，通过囊泡融合从高表达Trek-1的HEK细胞系获得的泡状融合形成。所得到的重组天然膜经过光漂白后通过荧光恢复在聚合物电活性换能器聚(3，4-乙二氧基噻吩聚苯乙烯磺酸)(PEDOT：PSS)的膜上形成可移动的双层。PEDOT：PSS电极随后用于定量电化学阻抗谱测量配体介导的Trek-1与两种已知分别抑制和激活Trek-1通道的化合物Spadin和花生四烯酸之间的相互作用。PEDOT：基于PSS的有机电化学晶体管随后被用于对Trek-1功能的光学和电子组合测量。由于膜集成装置的健壮性和获得的高定量电信号，该技术在未来跨膜蛋白调节器的高通量筛选中具有很高的前景。这与基于活细胞的电生理分析(例如，膜片钳)相比，后者在成本、可用性和与光学转导的兼容性方面都较差。
Transmembrane proteins represent a major target for modulating cell activity, both in terms of therapeutics drugs and for pathogen interactions. Work on screening such therapeutics or identifying toxins has been severely limited by the lack of available methods that would give high content information on functionality (ideally multimodal) and that are suitable for high-throughput. Here, we have demonstrated a platform that is capable of multimodal (optical and electronic) screening of ligand gated ion-channel activity in human-derived membranes. The TREK-1 ion-channel was expressed within supported lipid bilayers, formed via vesicle fusion of blebs obtained from the HEK cell line overexpressing TREK-1. The resulting reconstituted native membranes were confirmed via fluorescence recovery after photobleaching to form mobile bilayers on top of films of the polymeric electroactive transducer poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). PEDOT:PSS electrodes were then used for quantitative electrochemical impedance spectroscopy measurements of ligand-mediated TREK-1 interactions with two compounds, spadin and arachidonic acid, known to suppress and activate TREK-1 channels, respectively. PEDOT:PSS-based organic electrochemical transistors were then used for combined optical and electronic measurements of TREK-1 functionality. The technology demonstrated here is highly promising for future high-throughput screening of transmembrane protein modulators owing to the robust nature of the membrane integrated device and the highly quantitative electrical signals obtained. This is in contrast with live-cell-based electrophysiology assays (e.g., patch clamp) which compare poorly in terms of cost, usability, and compatibility with optical transduction.