Lee Sungmoo, Song Yoon-Kyu, Baker Bradley J
Center for Functional Connectomics, Brain Science Institute, Korea Institute of Science and Technology, Seoul, South Korea.
Program in Nano Science and Technology, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea.
Front Cell Neurosci. 2019 Oct 29;13:482. doi: 10.3389/fncel.2019.00482. eCollection 2019.
Genetically-encoded indicators of neuronal activity enable the labeling of a genetically defined population of neurons to optically monitor their activities. However, researchers often find difficulties in identifying relevant signals from excessive background fluorescence. A photoactivatable version of a genetically encoded calcium indicator, sPA-GCaMP6f is a good example of circumventing such an obstacle by limiting the fluorescence to a region of interest defined by the user. Here, we apply this strategy to genetically encoded voltage (GEVI) and pH (GEPI) indicators. Three photoactivatable GEVI candidates were considered. The first one used a circularly-permuted fluorescent protein, the second design involved a Förster resonance energy transfer (FRET) pair, and the third approach employed a pH-sensitive variant of GFP, ecliptic pHluorin. The candidate with a variant of ecliptic pHluorin exhibited photoactivation and a voltage-dependent fluorescence change. This effort also yielded a pH-sensitive photoactivatable GFP that varies its brightness in response to intracellular pH changes.
神经元活动的基因编码指示剂能够对基因定义的神经元群体进行标记，从而以光学方式监测它们的活动。然而，研究人员常常难以从过量的背景荧光中识别出相关信号。基因编码钙指示剂的光激活版本sPA-GCaMP6f就是一个很好的例子，它通过将荧光限制在用户定义的感兴趣区域来规避这一障碍。在此，我们将此策略应用于基因编码电压（GEVI）指示剂和pH（GEPI）指示剂。我们考虑了三种光激活GEVI候选物。第一种使用了环形排列的荧光蛋白，第二种设计涉及荧光共振能量转移（FRET）对，第三种方法采用了GFP的pH敏感变体——偏黄荧光蛋白。带有偏黄荧光蛋白变体的候选物表现出光激活和电压依赖性荧光变化。这项工作还产生了一种pH敏感的光激活GFP，它会根据细胞内pH变化改变其亮度。
