Experimental Biophysics, Humboldt-Universität zu Berlin, D-10115 Berlin, Germany.
Proc Natl Acad Sci U S A. 2011 May 3;108(18):7595-600. doi: 10.1073/pnas.1017210108. Epub 2011 Apr 19.
Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-to-cysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for light-controlled activation of neurons.
通道视紫红质-2（ChR2）已成为神经科学中不可或缺的工具，它可以通过短光脉冲精确地引发动作电位。对于许多光生理实验来说，限制因素是 ChR2 诱导的光电流相对较小。我们筛选了大量 ChR2 点突变体，发现第 159 位的苏氨酸突变为半胱氨酸后，光电流幅度显著增加。当我们在海马锥体神经元中测试 T159C 突变体时，在目前可用的通道视紫红质无法驱动尖峰的非常低的光强度下，可以诱导动作电位。生物物理特性表明，大多数 ChR2 变体在去极化膜电位下的动力学大大减慢。我们表明，最近发表的 E123T 取代消除了这种电压敏感性并加速了通道动力学。当我们将 T159C 与 E123T 结合使用时，所得的双突变体产生了快速、大振幅的光电流，并在广泛的频率范围内提高了单个动作电位诱导的精度，表明它可能成为光控神经元激活的标准。
