Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function
Spectral characteristics of the photocycle of channelrhodopsin-2 and its implication for channel function
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DOI：
10.1016/j.jmb.2007.10.072
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发表时间：
2008-01-18
影响因子：
5.6
通讯作者：
Bamberg, Ernst
中科院分区：
文献类型：
--
作者：
Bamann, Christian;Kirsch, Taryn;Bamberg, Ernst
在2003年，来自莱茵衣藻的channelrhodopsin-2（ChR 2）被发现是一种光门控阳离子通道，从那时起，该通道就成为了一种优秀的工具，可以通过光控制培养物中的神经元细胞，以及以非侵入性方式以高时间和空间分辨率控制活体动物。然而，很少有人知道的频谱特性和它们的关系的通道功能。我们在毕赤酵母中表达了ChR 2，并纯化了其蛋白。闪光光解数据结合膜片钳研究阐明光循环。该蛋白质在光激发前在类似于480 nm处吸收最大，并显示出闪光诱导的吸光度变化与至少两种不同的光中间体。四个弛豫过程可以提取的时间过程中，我们已经分析了在一个线性模型的光循环导致的动力学中间体P-1至P-4。在400 nm处的一个短寿命的光中间体，表明视网膜席夫碱的去质子化，随后是具有毫秒寿命的红移（520 nm）物种。光循环中的前三个动力学中间体，P-1至P-3，主要由红移520 nm的物质描述。400-mn物种对P-1和P-2的贡献较小。第四个，P-4，在光谱上几乎与基态相同，并持续到秒时间区。我们比较了光谱数据的电流测量全细胞膜片钳条件下的HEK 293细胞。光谱和电生理测定的中间体的寿命是非常一致的。中间体P-2和P-3（在520 nm处吸收）被鉴定为通道的阳离子渗透态。在稳定光下，观察到光电流被绿色光（540 nm）调制。我们的结论是，红移的光谱物种代表开放的通道状态，和这个中间体的热弛豫，从P-3到P-4的过渡，耦合到通道关闭。(c)2007年由Elsevier Ltd.出版
In 2003, channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii was discovered to be a light-gated cation channel, and since that time the channel became an excellent tool to control by light neuronal cells in culture as well as in living animals with high temporal and spatial resolution in a noninvasive manner. However, little is known about the spectral properties and their relation to the channel function. We have expressed ChR2 in the yeast Pichia pastoris and purified the protein. Flash-photolysis data were combined with patch-clamp studies to elucidate the photocycle. The protein absorbs maximally at similar to 480 nm before light excitation and shows flash-induced absorbance changes with at least two different photointermediates. Four relaxation processes can be extracted from the time course that we have analysed in a linear model for the photocycle leading to the kinetic intermediates P-1 to P-4. A short-lived photoi-ntermediate at 400 nm, suggesting a deprotonation of the retinal Schiff base, is followed by a redshifted (520 nm) species with a millisecond lifetime. The first three kinetic intermediates in the photocycle, P-1 to P-3, are described mainly by the redshifted 520-nm species. The 400-mn species contributes to a smaller extent to P-1 and P-2. The fourth one, P-4, is spectroscopically almost identical with the ground state and lasts into the seconds time region. We compared the spectroscopic data to current measurements under whole-cell patch-clamp conditions on HEK 293 cells. The lifetimes of the spectroscopically and electrophysiologically determined intermediates are in excellent agreement. The intermediates P-2 and P-3 (absorbing at 520 nm) are identified as the cation permeating states of the channel. Under stationary light, a modulation of the photocurrent by green light (540 nm) was observed. We conclude that the red-shifted spectral species represents the open channel state, and the thermal relaxation of this intermediate, the transition from P-3 to P-4, is coupled to channel closing. (c) 2007 Published by Elsevier Ltd.