Department of Neuroscience and Cell Biology, Montana State University, Bozeman, Montana, United States of America.
Montana Molecular, Bozeman, Montana, United States of America.
PLoS One. 2020 Dec 30;15(12):e0229051. doi: 10.1371/journal.pone.0229051. eCollection 2020.
Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.
基因编码荧光生物传感器是研究神经系统中复杂信号的有力工具，现在 Ca2+和电压传感器都可用于研究整个神经回路的信号行为。迫切需要改进传感器，但改进它们具有挑战性，因为测试它们涉及低通量、劳动密集型的过程。我们的目标是创建可通过短暂的蓝光脉冲激活的合成可兴奋细胞，作为下一代传感器筛选的高通量平台。在这个活细胞系统中，蓝光激活腺苷酸环化酶（bPAC），从而增加细胞内 cAMP（Stierl M 等人，2011 年）。反过来，cAMP 打开 cAMP 门控离子通道。这会产生缓慢的全细胞 Ca2+瞬变和电压变化。为了提高这些瞬变的速度，我们添加内向整流钾通道 Kir2.1、细菌电压门控钠通道 NAVROSD 和 Connexin-43。结果是一个高度可重复的高通量活细胞系统，可用于筛选电压和 Ca2+传感器。
