Kojima Keiichi, Watanabe Hiroshi C, Doi Satoko, Miyoshi Natsuki, Kato Misaki, Ishikita Hiroshi, Sudo Yuki
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8654, Japan.
Biophys Physicobiol. 2018 Sep 7;15:179-188. doi: 10.2142/biophysico.15.0_179. eCollection 2018.
Anion channelrhodopsin-2 (ACR2), a light-gated channel recently identified from the cryptophyte alga , exhibits anion channel activity with exclusive selectivity. In addition to its novel function, ACR2 has become a focus of interest as a powerful tool for optogenetics. Here we combined experimental and computational approaches to investigate the roles of conserved carboxylates on the anion transport activity of ACR2 in membrane. First, we replaced six conserved carboxylates with a neutral residue (i.e. E9Q, E56Q, E64Q, E159Q, E219Q and D230N), and measured anion transport activity using expression system. E159Q and D230N exhibited significantly lower anion transport activity compared with wild-type ACR2 (1/12~1/3.4), which suggests that E159 and D230 play important roles in the anion transport. Second, to explain its molecular aspects, we constructed a homology model of ACR2 based on the crystal structure of a cation channelrhodopsin (ChR). The model structure showed a cavity formed by four transmembrane helices (TM1, TM2, TM3 and TM7) similar to ChRs, as a putative anion conducting pathway. Although E159 is not located in the putative pathway, the model structure showed hydrogen bonds between E159 and R129 with a water molecule. D230 is located in the pathway near the protonated Schiff base (PSB) of the chromophore retinal, which suggests that there is an interaction between D230 and the PSB. Thus, we demonstrated the functional importance and the hypothetical roles of two conserved carboxylates, E159 and D230, in the anion transport activity of ACR2 in membrane.
阴离子通道视紫红质-2（ACR2）是最近从隐藻中鉴定出的一种光门控通道，具有独特选择性的阴离子通道活性。除了其新功能外，ACR2作为光遗传学的一种强大工具，已成为人们关注的焦点。在这里，我们结合实验和计算方法来研究保守羧酸盐对ACR2在膜中阴离子转运活性的作用。首先，我们将六个保守羧酸盐替换为中性残基（即E9Q、E56Q、E64Q、E159Q、E219Q和D230N），并使用表达系统测量阴离子转运活性。与野生型ACR2相比，E159Q和D230N的阴离子转运活性显著降低（1/12~1/3.4），这表明E159和D230在阴离子转运中起重要作用。其次，为了解释其分子层面问题，我们基于阳离子通道视紫红质（ChR）的晶体结构构建了ACR2的同源模型。模型结构显示由四个跨膜螺旋（TM1、TM2、TM3和TM7）形成的一个腔，类似于ChRs，作为一个假定的阴离子传导途径。尽管E159不在假定途径中，但模型结构显示E159和R129之间通过一个水分子形成氢键。D230位于发色团视黄醛的质子化席夫碱（PSB）附近的途径中，这表明D230与PSB之间存在相互作用。因此，我们证明了两个保守羧酸盐E159和D230在ACR2在膜中阴离子转运活性中的功能重要性和假定作用。
