Venkatachalam Veena, Cohen Adam E
Biophysics Program, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts.
Departments of Chemistry and Chemical Biology and Physics, Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts; Howard Hughes Medical Institute, Chevy Chase, Maryland.
Biophys J. 2014 Oct 7;107(7):1554-63. doi: 10.1016/j.bpj.2014.08.020.
To study the impact of neural activity on cellular physiology, one would like to combine precise control of firing patterns with highly sensitive probes of cellular physiology. Light-gated ion channels, e.g., Channelrhodopsin-2, enable precise control of firing patterns; green fluorescent protein-based reporters, e.g., the GCaMP6f Ca(2+) reporter, enable highly sensitive probing of cellular physiology. However, for most actuator-reporter combinations, spectral overlap prevents straightforward combination within a single cell. Here we explore multiwavelength control of channelrhodopsins to circumvent this limitation. The "stoplight" technique described in this article uses channelrhodopsin variants that are opened by blue light and closed by orange light. Cells are illuminated with constant blue light to excite fluorescence of a green fluorescent protein-based reporter. Modulated illumination with orange light negatively regulates activation of the channelrhodopsin. We performed detailed photophysical characterization and kinetic modeling of four candidate stoplight channelrhodopsins. The variant with the highest contrast, sdChR(C138S,E154A), enabled all-optical measurements of activity-induced calcium transients in cultured rat hippocampal neurons, although cell-to-cell variation in expression levels presents a challenge for quantification.
为了研究神经活动对细胞生理学的影响，人们希望将放电模式的精确控制与细胞生理学的高灵敏度探针相结合。光门控离子通道，例如通道视紫红质-2，能够实现对放电模式的精确控制；基于绿色荧光蛋白的报告基因，例如GCaMP6f钙离子报告基因，能够对细胞生理学进行高灵敏度探测。然而，对于大多数促动器-报告基因组合而言，光谱重叠阻碍了在单个细胞内进行直接组合。在此，我们探索通道视紫红质的多波长控制以克服这一限制。本文所述的“交通信号灯”技术使用的通道视紫红质变体可被蓝光开启并被橙光关闭。用恒定蓝光照射细胞以激发基于绿色荧光蛋白的报告基因的荧光。用橙光进行调制照射可对通道视紫红质的激活进行负调控。我们对四种候选交通信号灯通道视紫红质进行了详细的光物理表征和动力学建模。对比度最高的变体sdChR(C138S,E154A)，能够对培养的大鼠海马神经元中活动诱导的钙瞬变进行全光学测量，尽管表达水平的细胞间差异给定量分析带来了挑战。
