Studies on cell death and survival signals via connexin channels during cell injury

间隙连接被认为在调节细胞死亡和存活中起重要作用。基因敲除和RNA干扰等连接蛋白（Cxs）的遗传方法最终降低了整个细胞中的Cx水平，这些方法提供了直接证据，证明间隙连接细胞间通讯对细胞存活至关重要。然而，一种Cx可能会根据其位置和/或时间事件的特定阶段执行不同的功能。这种蛋白质功能的时空差异很难用传统的遗传方法来分析。在这项研究中，我们开发了多光子激发诱发的发色团辅助激光失活（MP-CALI），该方法可以通过短暂的激光照射使cx增强的绿色荧光蛋白（EGFP）形成的特定间隙连接在时空上失活。实验采用由Cx43-EGFP组成的小间隙连接斑块（长度<1 μm）连接一对HeLa细胞。使用双全细胞电压钳法测量更多的结电流，用850 nm飞秒激光照射Cx43-EGFP斑块的单点，激光辐照度为2.7 MW/cm^2，照射时间为380 ms。我们发现在激光照射过程中，缝隙结电流减小了80%。然而，同样的辐照并没有改变表达Cx43的HeLa细胞对之间的间隙连接电流，这表明850 nm波长的MP-CALI特异性地失活了Cx-EGFP形成的间隙连接。我们还发现MPCALI可以抑制特定细胞对之间的染料偶联。Cx43-EGFP的MP-CALI显示出缝隙结电流的减小与激光功率有关，即电流减小的阈值激光功率在1.0 ~ 1.3 MW/cm2之间。活性氧（ROS）清除剂依达拉屈和叠氮化钠可损害MP-CALI反应，表明ROS参与了MP-CALI。因此，MP-CALI可用于解析新的Cx函数。少

Gap junctions are considered to play an important role in moderating cell death and survival. Genetic approaches to the connexins (Cxs) such as gene knockouts and RNA interference, which ultimately reduce Cx levels in the entire cell, have provided direct evidence that gap junctional intercellular communication is essential for cell survival. However, it is possible that one kind of Cx performs different functions depending on its location and/or a particular stage of temporal events. Such spatiotemporal differences in protein function are difficult to analyze with conventional genetic approaches. In this study, we have developed multiphoton excitation-evoked chromophore-assisted laser inactivation (MP-CALI),that specific gap junctions made of Cx-enhanced green fluorescent protein(EGFP)can be spatiotemporally inactivated by brief laser irradiation. Experiments were performed on a pair of HeLa cells connected via a small gap junction plaque (length, <1 μm)consisting of Cx43-EGFP. While … More junctional currents were measured using the dual whole-cell voltage-clamp -method, a single point of the Cx43-EGFP plaque was irradiated with 850-nm femtosecond laser light with a laser irradiance of 2.7 MW/cm^2for 380 ms. We found that during the laser irradiation, the gap junction current decreased "○80%. However the same irradiation did not change gap junctional currents between HeLa cell pairs expressing Cx43 tagged with monomeric red fluorescent protein(mRFP),indicating that MP-CALI at a wavelength of 850 nm specifically inactivates gap junctions made of Cx-EGFP. We also found that MPCALI can inhibit dye coupling between specifically selected cell pairs. MP-CALI of Cx43-EGFP showed a laser power dependent decrease of gap junction current i.e., the threshold laser power for current reduction is between 1.0 and 1.3 MW/cm2. Reactive oxygen species (ROS) scavengers, edaravone and sodium azide, impaired the MP-CALI response, indicating involvement of ROS in MP-CALI. Thus, MP-CALI could be useful for the elucidation of new Cx functions. Less