The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity.
The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity.
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DOI：
10.1038/srep43358
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发表时间：
2017-03-03
影响因子：
4.6
通讯作者：
Spudich JL
中科院分区：
文献类型：
--
作者：
Govorunova EG;Sineshchekov OA;Rodarte EM;Janz R;Morelle O;Melkonian M;Wong GK;Spudich JL
在隐芽藻中发现的天然阴离子通道视紫红质(ACRs)在Cl-−的能斯特平衡电位以上的膜电位处产生大的超极化电流，因此可作为光遗传学的有效抑制工具。我们在不同的隐芽植物中鉴定和鉴定了新的ACR同源物，表明它们都是阴离子选择性的，从而将该蛋白家族扩大到20个功能确认的成员。对阳离子通道视紫红质(CCR)的天然ACRs和工程Cl−传导突变体的序列比较表明，它们的阴离子选择性过滤器存在根本性的差异。特别是，通道视紫红质2中的Glu90残基在所有已鉴定的ACR中都是保守的，需要突变为中性或碱性残基才能赋予CCR阴离子选择性。新的ACR显示其光电流的幅度、动力学和光谱灵敏度有很大的变化。一种值得注意的变种，命名为“ZipACR”，在抑制光遗传学方面特别有希望，因为它结合了比以前报道的ACR更大的电流幅度和前所未有的快速电导周期(电流半衰期取决于电压2-4 ms)。在培养的小鼠海马神经元中表达的ZipACR能够精确地光抑制高达50 Hz频率的单个棘波。
Natural anion channelrhodopsins (ACRs) discovered in the cryptophyte alga Guillardia theta generate large hyperpolarizing currents at membrane potentials above the Nernst equilibrium potential for Cl− and thus can be used as efficient inhibitory tools for optogenetics. We have identified and characterized new ACR homologs in different cryptophyte species, showing that all of them are anion-selective, and thus expanded this protein family to 20 functionally confirmed members. Sequence comparison of natural ACRs and engineered Cl−-conducting mutants of cation channelrhodopsins (CCRs) showed radical differences in their anion selectivity filters. In particular, the Glu90 residue in channelrhodopsin 2, which needed to be mutated to a neutral or alkaline residue to confer anion selectivity to CCRs, is nevertheless conserved in all of the ACRs identified. The new ACRs showed a large variation of the amplitude, kinetics, and spectral sensitivity of their photocurrents. A notable variant, designated “ZipACR”, is particularly promising for inhibitory optogenetics because of its combination of larger current amplitudes than those of previously reported ACRs and an unprecedentedly fast conductance cycle (current half-decay time 2–4 ms depending on voltage). ZipACR expressed in cultured mouse hippocampal neurons enabled precise photoinhibition of individual spikes in trains of up to 50 Hz frequency.