Doi Satoko, Mori Arisa, Tsukamoto Takashi, Reissig Louisa, Ihara Kunio, Sudo Yuki
Division of Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Japan.
Photochem Photobiol Sci. 2015 Sep 26;14(9):1628-36. doi: 10.1039/c5pp00213c. Epub 2015 Jun 22.
Channelrhodopsins have become a focus of interest because of their ability to control neural activity by light, used in a technology called optogenetics. The channelrhodopsin in the eukaryote Chlamydomonas reinhardtii (CrChR-1) is a light-gated cation channel responsible for motility changes upon photo-illumination and a member of the membrane-embedded retinal protein family. Recent crystal structure analysis revealed that CrChR-1 has unique extended modules both at its N- and C-termini compared to other microbial retinal proteins. This study reports the first successful expression of a ChR-1 variant in Escherichia coli as a holoprotein: the ChR-1 variant lacking both the N- and C-termini (CrChR-1_82-308). However, compared to ChR-1 having the extended modules (CrChR-1_1-357), truncation of the termini greatly altered the absorption maximum and photochemical properties, including the pKa values of its charged residues around the chromophore, the reaction rates in the photocycle and the photo-induced ion channeling activity. The results of some experiments regarding ion transport activity suggest that CrChR-1_82-308 has a proton channeling activity even in the dark. On the basis of these results, we discuss the structural and functional roles of the N- and C-terminal extended modules in CrChR-1.
由于能够通过光来控制神经活动，通道视紫红质已成为人们关注的焦点，这种技术被称为光遗传学。真核生物莱茵衣藻（CrChR-1）中的通道视紫红质是一种光门控阳离子通道，负责光照后运动性的变化，是膜嵌入视网膜蛋白家族的成员。最近的晶体结构分析表明，与其他微生物视网膜蛋白相比，CrChR-1在其N端和C端均具有独特的延伸模块。本研究报道了ChR-1变体在大肠杆菌中作为全蛋白的首次成功表达：缺失N端和C端的ChR-1变体（CrChR-1_82-308）。然而，与具有延伸模块的ChR-1（CrChR-1_1-357）相比，末端的截短极大地改变了最大吸收和光化学性质，包括发色团周围带电残基的pKa值、光循环中的反应速率以及光诱导离子通道活性。一些关于离子转运活性的实验结果表明，CrChR-1_82-308即使在黑暗中也具有质子通道活性。基于这些结果，我们讨论了CrChR-1中N端和C端延伸模块的结构和功能作用。
