Lin John Y, Lin Michael Z, Steinbach Paul, Tsien Roger Y
Department of Pharmacology, University of California, San Diego, California, USA.
Biophys J. 2009 Mar 4;96(5):1803-14. doi: 10.1016/j.bpj.2008.11.034.
Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile(170) of ChEF to Val (yielding "ChIEF") accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.
通道视紫红质2（ChR2）是一种来自莱茵衣藻的光激活非选择性阳离子通道，已成为一种用于激发转染该通道的神经元的有用工具。衣藻的另一种通道视紫红质ChR1因其质子选择性通透性而较少受到关注。通过构建ChR1和ChR2跨膜结构域的嵌合体，并结合定点诱变，我们开发了一种通道视紫红质变体，命名为ChEF，它在持续光刺激期间的失活明显较少。与ChR2的77%失活相比，ChEF仅经历33%的失活。将ChEF的Ile(170)点突变为Val（产生“ChIEF”）可加速通道关闭速率，同时保持较低的失活率，从而在HEK293细胞和培养的海马神经元中以高于25 Hz的频率刺激时产生更一致的反应。此外，这些变体具有改变的光谱响应、光敏感性和通道选择性。ChEF和ChIEF允许对去极化进行更精确的时间控制，并能诱导更接近自然放电模式的动作电位序列。
