Govorunova Elena G, Sineshchekov Oleg A, Li Hai, Gou Yueyang, Chen Hongmei, Yang Shuyuan, Wang Yumei, Mitchell Stephen, Palmateer Alyssa, Brown Leonid S, St-Pierre François, Xue Mingshan, Spudich John L
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston McGovern Medical School, Houston, United States.
The Cain Foundation Laboratories, Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, United States.
Elife. 2025 Sep 19;14:RP106508. doi: 10.7554/eLife.106508.
Light-gated ion channels from protists (channelrhodopsins or ChRs) are optogenetic tools widely used for controlling neurons and cardiomyocytes. Multiplex optogenetic applications require spectrally separated molecules, which are difficult to engineer without disrupting channel function. Scanning numerous sequence databases, we identified three naturally blue-shifted ChRs from ancyromonads. They form a separate branch on the phylogenetic tree and contain residue motifs characteristic of anion ChRs (ACRs). However, only two conduct chloride, whereas the closely related homolog generates inward cation currents in mammalian cells under physiological conditions, significantly exceeding those by previously known tools with similar spectral maxima (peak absorption at ~440 nm). Measurements of transient absorption changes and pH titration of purified proteins combined with mutant analysis revealed the roles of the residues in the photoactive site. Ancyromonad ChRs could be activated by near-infrared two-photon illumination, a technique that enables the deeper-tissue optogenetic activation of specific neurons in three dimensions. Both ancyromonad ACRs allowed optogenetic silencing of mouse cortical neurons in brain slices. ACR (ACR) expression in cholinergic neurons enabled photoinhibition of pharyngeal muscle contraction in live worms. Overall, our results deepen the mechanistic understanding of light-gated channel function and expand the optogenetic toolkit with potent, blue-shifted ChRs.
来自原生生物的光门控离子通道（视紫红质通道蛋白或ChRs）是广泛用于控制神经元和心肌细胞的光遗传学工具。多重光遗传学应用需要光谱分离的分子，而在不破坏通道功能的情况下很难对其进行工程改造。通过扫描众多序列数据库，我们从锚定单胞菌中鉴定出三种天然蓝移的ChRs。它们在系统发育树上形成一个单独的分支，并含有阴离子ChRs（ACRs）的特征性残基基序。然而，只有两种能传导氯化物，而与之密切相关的同系物在生理条件下在哺乳动物细胞中产生内向阳离子电流，显著超过那些具有相似光谱最大值（峰值吸收在~440nm）的先前已知工具所产生的电流。对纯化蛋白的瞬态吸收变化和pH滴定测量结合突变分析揭示了光活性位点中残基的作用。锚定单胞菌ChRs可通过近红外双光子照射激活，这是一种能够在三维空间中对特定神经元进行更深组织光遗传学激活的技术。两种锚定单胞菌ACRs都能使脑片中的小鼠皮层神经元实现光遗传学沉默。胆碱能神经元中ACR的表达能够对活蠕虫咽部肌肉收缩进行光抑制。总体而言，我们的结果加深了对光门控通道功能的机制理解，并用强大的蓝移ChRs扩展了光遗传学工具包。
