Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277-8564 Japan.
Sci Rep. 2017 Feb 2;7:41879. doi: 10.1038/srep41879.
Anion channelrhodopsin-2 (ACR2) was recently identified from the cryptophyte algae Guillardia theta and has become a focus of interest in part because of its novel light-gated anion channel activity and its extremely high neural silencing activity. In this study, we tried to express ACR2 in Escherichia coli cells as a recombinant protein. The E. coli cells expressing ACR2 showed an increase in pH upon blue-light illumination in the presence of monovalent anions and the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), indicating an inward anion channel activity. Then, taking advantage of the E. coli expression system, we performed alanine-scanning mutagenesis on conserved basic amino acid residues. One of them, R84A, showed strong signals compared with the wild-type, indicating an inhibitory role of R84 on Cl transportation. The signal was strongly enhanced in R84E, whereas R84K was less effective than the wild-type (i.e., R84). These results suggest that the positive charge at position 84 is critical for the inhibition. Thus we succeeded in functional expression of ACR2 in E. coli and found the inhibitory role of R84 during the anion transportation.
阴离子通道视紫红质 2（ACR2）最近从隐藻藻类中鉴定出来，因其新颖的光门阴离子通道活性和极高的神经沉默活性而成为研究热点。在这项研究中，我们试图在大肠杆菌细胞中表达 ACR2 作为重组蛋白。在存在单价阴离子和质子载体羰基氰化物 m-氯苯腙（CCCP）的情况下，表达 ACR2 的大肠杆菌细胞在蓝光照射下显示出 pH 值升高，表明存在内向阴离子通道活性。然后，我们利用大肠杆菌表达系统对保守的碱性氨基酸残基进行丙氨酸扫描诱变。其中一个，R84A，与野生型相比显示出较强的信号，表明 R84 对 Cl 运输具有抑制作用。R84E 的信号增强，而 R84K 的效果不如野生型（即 R84）。这些结果表明，位置 84 的正电荷对抑制作用至关重要。因此，我们成功地在大肠杆菌中实现了 ACR2 的功能表达，并发现了 R84 在阴离子运输过程中的抑制作用。
