Human PIEZO1: Removing Inactivation
Human PIEZO1: Removing Inactivation
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DOI：
10.1016/j.bpj.2013.07.019
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发表时间：
2013-08-20
影响因子：
3.4
通讯作者：
Sachs, Frederick
中科院分区：
文献类型：
--
作者：
Bae, Chilman;Gottlieb, Philip A.;Sachs, Frederick
PIEZO 1是一种失活的真核阳离子选择性机械敏感离子通道。通道中有两个位点，当单独突变时，通过减慢失活导致干细胞性贫血。通过在两个位点引入突变，一个与干细胞症相关，另一个是人工的，我们能够消除失活。双突变体（DhPIEZO 1）具有蛋氨酸（M2225 R）和精氨酸（R2456 K）的赖氨酸-e取代。失活的损失是伴随着类似的30毫米汞柱的移动的激活曲线，以较低的压力和较慢的失活率。门控的斜率灵敏度对于野生型和突变体是相同的，表明闭合和开放状态之间的尺寸变化不受突变的影响。单一通道电导不受突变的影响，因此这些位点与孔无关。DhPIEZO 1可逆抑制肽GsMTx 4作为门控修饰剂。使用复杂的刺激波形解决通道动力学，并将数据拟合到详细平衡的三态回路。该反应有两个压力依赖性速率，关闭到打开和失活到关闭。开启速率的压力敏感性与关闭速率的不敏感性意味着它们之间的能量势垒位于开启状态附近。失活突变周期分析表明，这两个网站强烈相互作用，即使他们被假定为在膜的两侧。
PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginin-e for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by similar to 30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.