Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization.
Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization.
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DOI：
10.1016/j.neuron.2017.03.039
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发表时间：
2017-04-19
期刊：
影响因子：
16.2
通讯作者：
Patapoutian A
中科院分区：
文献类型：
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作者：
Dubin AE;Murthy S;Lewis AH;Brosse L;Cahalan SM;Grandl J;Coste B;Patapoutian A
表征机械激活 (MA) 电流的黄金标准是通过初始细胞中候选通道的异源表达。最近的两项研究描述了使用这种范式的 MA 通道。 TMEM150c 被提议作为 MA 通道的一个组成部分，部分基于异源表达方法。在另一项研究中，基于机械不敏感 ASIC1 通道的成孔结构域和 Piezo1 之间嵌合体的表达，Piezo1 的 N 端“螺旋桨”结构域被提议构成一个内在的机械敏感模块。当我们试图复制这些结果时，我们发现每个构建体在一小部分幼稚 HEK 细胞中赋予适度的 MA 电流，与已发表的工作类似。引人注目的是，在内源性 Piezo1 被 CRISPR/Cas9 失活的细胞中未检测到这些 MA 电流。这些结果强调了选择缺乏内源 MA 通道的细胞来测定各种蛋白质的机械转导特性的重要性。
A gold standard for characterizing mechanically-activated (MA) currents is via heterologous expression of candidate channels in naïve cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of a MA channel partly based on a heterologous expression approach. In another study, Piezo1’s N-terminal “propeller” domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechano-insensitive ASIC1 channel and Piezo1. When we attempted to replicate these results we found each construct conferred modest MA currents in a small fraction of naïve HEK cells similar to the published work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9-inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins.