Center for Single Molecule Biophysics, Department of Physiology and Biophysics, 301 Cary Hall, State University of New York, Buffalo, New York 14214, USA.
Biochemistry. 2011 Jul 26;50(29):6295-300. doi: 10.1021/bi200770q. Epub 2011 Jun 29.
Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ∼30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.
细胞可以通过门控机械敏感离子通道（MSCs）来响应机械应激。Piezo1 的克隆，一种真核阳离子选择性 MSC，定义了一个用于在细胞水平上研究机械转导的新系统。因为 Piezo1 具有类似于选择性地被肽 GsMTx4 抑制的内源性阳离子 MSCs 的电生理特性，所以我们测试了该肽是否靶向 Piezo1 活性。在微摩尔浓度的细胞外 GsMTx4 可逆地抑制了转染的 HEK293 细胞的外面向外斑片中约 80%的机械诱导电流。抑制是电压不敏感的，与内源性 MSCs 一样，镜像 d 对映体的抑制作用与 l 相似。基于 Piezo1 电流动力学的结合和离解速率常数提供了 7.0×10(5)M(-1)s(-1)和 0.11s(-1)的结合和离解速率，以及 K(D)约为 155nM，与之前报道的内源性 MSCs 值相似。与预测的门控修饰剂行为一致，GsMTx4 使压力门控曲线向右移动约 30mmHg，并在关闭的通道上起作用。相比之下，链霉素，一种阳离子 MSCs 的非特异性抑制剂，显示出与开放通道阻塞有关的使用依赖性抑制特征。该肽没有阻断外面向外斑中的机械通道 TREK-1 的电流。全细胞 Piezo1 电流也被 GsMTx4 可逆地抑制，尽管离解速率几乎与外面向外斑相同，但观察到的是开放通道阻断的进入速率。GsMTx4 靶向 Piezo1 的机械敏感性的能力支持该通道在药理学试剂和诊断测定的高通量筛选中的应用。
