Effect of Redox-Modifying Agents on the Activity of Channelrhodopsin-2.
Effect of Redox-Modifying Agents on the Activity of Channelrhodopsin-2.
复制标题
氧化还原修饰剂对通道视紫红质 2 活性的影响。
DOI：
10.1111/cns.12662
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发表时间：
2017-03
影响因子：
5.5
通讯作者：
Xiong ZG
中科院分区：
文献类型：
--
作者：
Wu BM;Leng TD;Inoue K;Li J;Xiong ZG
近年来，藻蛋白ChR 2被广泛应用于光遗传学技术中，通过对遗传定义的神经元进行微创和时间精确的光刺激来研究复杂神经元网络的功能。然而，与任何其他新技术一样，目前的光遗传方法具有各种限制。此外，ChR 2如何响应与各种生理/病理条件相关的复杂生化变化，在很大程度上是未知的。在目前的研究中，我们研究了细胞氧化还原状态的变化是否会影响ChR 2通道的活性。采用全细胞膜片钳技术研究了还原剂和氧化剂对蓝光激活的ChR 2电流的影响。我们发现，还原剂二硫苏糖醇（DTT）显着增强的ChR 2电流在一个可逆的和浓度依赖性的方式。内源性还原剂谷氨酰胺对ChR 2电流也有类似的作用。氧化剂5，5 '-二硫代-双-（2-硝基苯甲酸）（DTNB）本身对ChR 2电流无影响，但可完全逆转DTT的增强作用。DTT还导致电流-电压关系偏移23±4.31 mV，表明离子选择性发生变化。综上所述，这些数据表明，ChR 2的氧化还原修饰在其对光刺激的敏感性中起着重要作用。我们的研究结果不仅有助于更好地理解ChR 2在氧化还原状态变化常见的生理/病理条件下的行为，而且还为进一步优化这一重要的视蛋白提供了新的方向。
The algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown. In the current study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels. Whole cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light. We show that the reducing agent Dithiothreitol (DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) has no effect on ChR2 currents by itself, however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current-voltage relationship by 23±4.31 mV, suggesting a change in ion selectivity. Taken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin.