Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.
Cation and Anion Channelrhodopsins: Sequence Motifs and Taxonomic Distribution.
复制标题
DOI：
10.1128/mbio.01656-21
复制
发表时间：
2021-08-31
期刊：
影响因子：
6.4
通讯作者：
Spudich JL
中科院分区：
文献类型：
--
作者：
Govorunova EG;Sineshchekov OA;Li H;Wang Y;Brown LS;Palmateer A;Melkonian M;Cheng S;Carpenter E;Patterson J;Wong GK;Spudich JL
阳离子通道视紫红质（CCRs）和阴离子通道视紫红质（ACRs）主要来自莱茵衣藻（Chlamydomonas reinhardtii）和吉拉迪亚（Guillardia theta）两种藻类，已被广泛用作光遗传学工具，利用光控制细胞膜电位。我们挖掘了藻类和其他原生生物的多核苷酸测序项目和宏基因组样本，从四个通道视紫红质家族中鉴定了75个通道视紫红质同源物，其中包括本研究中在鞭毛藻中发现的一个。我们对33个来自不同系统发育谱系的天然通道紫红质变体和10个宏基因组同源物进行了电生理分析，以寻找通道紫红质离子选择性、光电流脱敏和光谱调谐的序列决定因素。我们的研究结果表明，电导路径附近谷氨酸盐数量减少与阴离子选择性的关联取决于更广泛的蛋白质背景，因为具有与隐生植物acr相同的谷氨酸模式的prasinophyte同源物具有阳离子选择性。脱敏也广泛依赖于环境，如在层堆acr的一个分支及其宏基因组同源物中，其程度大致与其序列的系统发育关系相关。在光谱调谐方面，我们鉴定了两个红移光谱至585 nm的葡萄树ccr。它们在视网膜结合口袋中表现出第三种残基模式，与已知的仅有的两种红移通道视紫红质（即，CCR crimson和RubyACRs）明显不同。在隐生植物ACRs中，我们在视网膜结合袋中确定了三个特定的残基位置，这些残基位置决定了其光谱最大值的波长。最后，我们发现在第三跨膜螺旋中具有TCP基序和宏基因组同源物的鞭毛藻紫红质具有通道活性。
Cation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from four channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with a glutamate pattern identical to that in cryptophyte ACRs are cation selective. Desensitization is also broadly context dependent, as in one branch of stramenopile ACRs and their metagenomic homologs, its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, we identified two prasinophyte CCRs with red-shifted spectra to 585 nm. They exhibit a third residue pattern in their retinal-binding pockets distinctly different from those of the only two types of red-shifted channelrhodopsins known (i.e., the CCR Chrimson and RubyACRs). In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity.