Center for Single Molecule Biophysics, Physiology and Biophysics, State University of New York at Buffalo, Buffalo, NY, USA.
Channels (Austin). 2012 Jul-Aug;6(4):282-9. doi: 10.4161/chan.21064. Epub 2012 Jul 1.
Piezo1 is a eukaryotic cation-selective mechanosensitive ion channel. To understand channel function in vivo, we first need to analyze and compare the response in the whole cell and the patch. In patches, Piezo1 inactivates and the current is fit well by a 3-state model with a single pressure-dependent rate. However, repeated stimulation led to an irreversible loss of inactivation. Remarkably, the loss of inactivation did not occur on a channel-by-channel basis but on all channels at the same time. Thus, the channels are in common mechanical domain. Divalent ions decreased the unitary conductance from ~68 pS to ~37 pS, irrespective of the cation species. Mg and Ca did not affect inactivation rates, but Zn caused a 3-fold slowing. CytochalasinD (cytoD) does not alter inactivation rates or the transition to the non-inactivating mode but does reduce the steady-state response. Whole-cell currents were similar to patch currents but also had significant differences. In contrast to the patch, cytoD inhibited the current suggesting that the activating forces were transmitted through the actin cytoskeleton. Hypotonic swelling that prestressed the cytoskeleton and the bilayer greatly increased the sensitivity of both control and cytoD cells so there are two pathways to transmit force to the channels. In contrast to patch, removing divalent ions decreased the whole-cell current. The difference between whole cell and patch properties provide new insights into our understanding of the Piezo1 gating mechanisms and cautions against generalization to in situ behavior.
Piezo1 是一种真核生物阳离子选择性机械敏感离子通道。为了理解通道在体内的功能，我们首先需要分析和比较全细胞和膜片钳中的响应。在膜片钳中，Piezo1 失活，电流可以通过一个具有单一压力依赖性速率的三状态模型很好地拟合。然而，重复刺激会导致不可逆的失活损失。值得注意的是，失活的损失不是逐个通道发生，而是所有通道同时发生。因此，这些通道处于共同的机械域。二价离子使单通道电导从约 68 pS 降低至约 37 pS，而与阳离子种类无关。Mg 和 Ca 不影响失活速率，但 Zn 导致 3 倍的失活减慢。细胞松弛素 D（cytoD）不改变失活速率或向非失活模式的转变，但会降低稳态响应。全细胞电流与膜片钳电流相似，但也有显著差异。与膜片钳不同，cytoD 抑制电流，表明激活力通过肌动球蛋白细胞骨架传递。使细胞骨架和双层预先紧张的低渗肿胀极大地增加了对照和 cytoD 细胞的敏感性，因此有两种途径将力传递到通道。与膜片钳不同，去除二价离子会降低全细胞电流。全细胞和膜片钳特性之间的差异为我们理解 Piezo1 门控机制提供了新的见解，并提醒我们不要将其推广到原位行为。
