Department of Physiology and Biophysics, State University of New York, Buffalo, NY, USA.
Biophys J. 2013 Aug 20;105(4):880-6. doi: 10.1016/j.bpj.2013.07.019.
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 arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼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.
PIEZO1 是一种失活的真核阳离子选择性机械敏感离子通道。已经定位了两个位点，当它们单独突变时，通过减缓失活导致干燥细胞性贫血。通过在两个位点引入突变，一个与干燥细胞有关，另一个是人为的，我们能够消除失活。双突变体（DhPIEZO1）具有精氨酸取代蛋氨酸（M2225R）和精氨酸取代赖氨酸（R2456K）的取代。失活的丧失伴随着激活曲线向较低压力和较慢失活速率的约 30mmHg 转移。门控的斜率敏感性对于野生型和突变体是相同的，表明在关闭和打开状态之间的尺寸变化不受突变的影响。单位通道电导不受突变影响，因此这些位点与孔无关。DhPIEZO1 可被肽 GsMTx4 可逆抑制，肽 GsMTx4 作为门控修饰剂起作用。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循环中。该反应具有两个压力依赖性速率，从关闭到打开和从失活到关闭。打开速率没有关闭速率的压力敏感性意味着它们之间的能量屏障位于打开状态附近。失活的突变体循环分析表明，两个位点相互作用很强，尽管它们被假定位于膜的相对侧。
