Characterization of Engineered Channel rhodopsin Variants with Improved Properties and Kinetics
Characterization of Engineered Channel rhodopsin Variants with Improved Properties and Kinetics
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DOI：
10.1016/j.bpj.2008.11.034
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发表时间：
2009-03-04
影响因子：
3.4
通讯作者：
Tsien, Roger Y.
中科院分区：
文献类型：
--
作者：
Lin, John Y.;Lin, Michael Z.;Tsien, Roger Y.
嗜铬视紫红质2（ChR2）是来自莱茵衣藻的一种光激活的非选择性阳离子通道，已成为激活其转染的神经元的有用工具。衣原体的另一种ChR，ChR1，由于其质子选择性渗透性而引起较少的关注。通过使ChR1和ChR2的跨膜结构域的嵌合体，结合定点诱变，我们开发了一种ChR变体，命名为ChEF，其在持续光刺激期间表现出显著较少的失活。ChEF仅经历33%的失活，而ChR2为77%。ChEF的Ile（170）到瓦尔的点突变（产生“ChIEF”）加速通道关闭的速率，同时保持减少的失活，当在HEK 293细胞和培养的海马神经元中以高于25 Hz刺激时，导致更一致的响应。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允许更精确的去极化时间控制，并可以诱导更接近自然尖峰模式的动作电位序列。
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.