A Blue-shifted Light-driven Proton Pump for Neural Silencing
A Blue-shifted Light-driven Proton Pump for Neural Silencing
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DOI：
10.1074/jbc.m113.475533
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发表时间：
2013-07-12
影响因子：
4.8
通讯作者：
Hayashi, Shigehiko
中科院分区：
文献类型：
--
作者：
Sudo, Yuki;Okazaki, Ayako;Hayashi, Shigehiko
离子转运视紫红质被广泛用作光诱导神经激活和沉默的光遗传学工具。研究最多的代表是细菌视紫红质（BR），其吸收绿色/红色光（类似于570 nm）并起到质子泵的作用。在光激发时，BR诱导跨膜的超极化，如果将其并入神经细胞，则导致其神经沉默。在这项研究中，我们表明，视网膜发色团周围的几个残基，这是完全保守的BR同源物之间的古细菌，参与光谱调谐的BR同源物（HwBR）和组合突变导致一个大的光谱蓝移（λ（最大值）= 498 nm），同时保持强大的泵浦活动。量子力学/分子力学计算显示，与野生型相比，突变体中发色团的β-紫罗兰酮环旋转了近似130度，这是因为在retinal的甲基和突变残基之间缺乏空间位阻，导致retinal的多烯链上的π共轭系统断裂。通过相同的突变，在另一种BR同系物archearhodopsin-3（也称为Arch）中也观察到类似的光谱蓝移。在秀丽隐杆线虫的神经细胞中成功表达了archearhodopsin-3的颜色变体，并且用蓝光（500 nm）照射导致蠕虫有效的运动麻痹。因此，我们成功地制造了用于神经沉默的蓝移质子泵。
Ion-transporting rhodopsins are widely utilized as optogenetic tools both for light-induced neural activation and silencing. The most studied representative is Bacteriorhodopsin (BR), which absorbs green/red light (similar to 570 nm) and functions as a proton pump. Upon photoexcitation, BR induces a hyperpolarization across the membrane, which, if incorporated into a nerve cell, results in its neural silencing. In this study, we show that several residues around the retinal chromophore, which are completely conserved among BR homologs from the archaea, are involved in the spectral tuning in a BR homolog (HwBR) and that the combination mutation causes a large spectral blue shift (lambda(max) = 498 nm) while preserving the robust pumping activity. Quantum mechanics/molecular mechanics calculations revealed that, compared with the wild type, the beta-ionone ring of the chromophore in the mutant is rotated similar to 130 degrees because of the lack of steric hindrance between the methyl groups of the retinal and the mutated residues, resulting in the breakage of the pi conjugation system on the polyene chain of the retinal. By the same mutations, similar spectral blue shifts are also observed in another BR homolog, archearhodopsin-3 (also called Arch). The color variant of archearhodopsin-3 could be successfully expressed in the neural cells of Caenorhabditis elegans, and illumination with blue light (500 nm) led to the effective locomotory paralysis of the worms. Thus, we successfully produced a blue-shifted proton pump for neural silencing.