Sequential absorption of two photons creates a bistable form of RubyACR responsible for its strong desensitization.
Sequential absorption of two photons creates a bistable form of RubyACR responsible for its strong desensitization.
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DOI：
10.1073/pnas.2301521120
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发表时间：
2023-05-23
影响因子：
11.1
通讯作者：
Spudich, John L.
中科院分区：
文献类型：
--
作者：
Sineshchekov, Oleg A.;Govorunova, Elena G.;Li, Hai;Wang, Yumei;Spudich, John L.
神经元活动的光遗传控制需要吸收更深穿透红光的工具。阴离子通道视紫红质（RubyACRs）是迄今为止红移最大的通道视紫红质。它们产生大的被动电流，但表现出快速和持久的脱敏，这损害了它们作为光遗传沉默剂的效用。这项研究揭示了RubyACR脱敏的光化学机制，以前没有描述任何其他通道视紫红质。具有红移吸收的类视紫红质在自然界中是罕见的，对于光遗传学是高度期望的，因为较长波长的光更深地穿透生物组织。RubyACRs（AnionalcohelRhodopsins）是一组来自破囊壶菌原生生物的四种密切相关的阴离子传导通道视紫红质，是已知的最红移的通道视紫红质，其最大吸收波长可达610 nm。它们的光电流很大，这是典型的蓝色和绿色吸收ACR，但它们在连续照明（脱敏）期间迅速下降，在黑暗中恢复非常缓慢。在这里，我们表明，持久的脱敏RubyACRs的结果从光化学没有观察到在任何以前研究的通道视紫红质。在640 nm处具有最大吸收的光循环中间体（P640）对第二光子的吸收使得RubyACR具有光致发光（即，在两种光谱不同的形式之间非常缓慢地相互转换）。这种形式的光循环涉及长寿命的非导电状态（Llong和Mlong），其形成是RubyACR光电流长期脱敏的原因。Llong和Mlong都是光活性的，并且分别在蓝色或紫外（UV）照射时转化为初始未光解状态。我们表明，可以减少或甚至消除通过使用纳秒激光闪光，列车的短光脉冲，而不是连续照明，以避免形成的Llong和Mlong，或通过应用蓝光脉冲之间的红光光转换Llong的初始非光解状态的脱敏。
Optogenetic control of neuronal activity requires tools that absorb deeper penetrating red light. Anion-conducting channelrhodopsins known as RubyACRs (Anion ChannelRhodopsins) are the most red-shifted channelrhodopsins to date. They generate large passive currents but show rapid and long-lasting desensitization, which compromises their utility as optogenetic silencers. This study reveals the photochemical mechanism of RubyACR desensitization not previously described for any other channelrhodopsin. Channelrhodopsins with red-shifted absorption, rare in nature, are highly desired for optogenetics because light of longer wavelengths more deeply penetrates biological tissue. RubyACRs (Anion ChannelRhodopsins), a group of four closely related anion-conducting channelrhodopsins from thraustochytrid protists, are the most red-shifted channelrhodopsins known with absorption maxima up to 610 nm. Their photocurrents are large, as is typical of blue- and green-absorbing ACRs, but they rapidly decrease during continuous illumination (desensitization) and extremely slowly recover in the dark. Here, we show that long-lasting desensitization of RubyACRs results from photochemistry not observed in any previously studied channelrhodopsins. Absorption of a second photon by a photocycle intermediate with maximal absorption at 640 nm (P640) renders RubyACR bistable (i.e., very slowly interconvertible between two spectrally distinct forms). The photocycle of this bistable form involves long-lived nonconducting states (Llong and Mlong), formation of which is the reason for long-lasting desensitization of RubyACR photocurrents. Both Llong and Mlong are photoactive and convert to the initial unphotolyzed state upon blue or ultraviolet (UV) illumination, respectively. We show that desensitization of RubyACRs can be reduced or even eliminated by using ns laser flashes, trains of short light pulses instead of continuous illumination to avoid formation of Llong and Mlong, or by application of pulses of blue light between pulses of red light to photoconvert Llong to the initial unphotolyzed state.
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影响因子：
62.1
作者：
Ernst, Oliver P.;Lodowski, David T.;Elstner, Marcus;Hegemann, Peter;Brown, Leonid S.;Kandori, Hideki
通讯作者：
Kandori, Hideki
影响因子：
7.7
作者：
Andrei AR;Debes S;Chelaru M;Liu X;Rodarte E;Spudich JL;Janz R;Dragoi V
通讯作者：
Dragoi V
影响因子：
16.6
作者：
Govorunova EG;Sineshchekov OA;Li H;Spudich JL
通讯作者：
Spudich JL
影响因子：
64.8
作者：
Wilson DE;Scholl B;Fitzpatrick D
通讯作者：
Fitzpatrick D
影响因子：
3.8
作者：
Dioumaev, AK
通讯作者：
Dioumaev, AK