An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli

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 Escheric...

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Bibliographic Details
Published in:Scientific reports 2017-02, Vol.7 (1), p.41879-41879, Article 41879
Main Authors: Doi, Satoko, Tsukamoto, Takashi, Yoshizawa, Susumu, Sudo, Yuki
Format: Article
Language:English
Subjects:
631/45/612/1237
631/57/1464
631/57/2270/1140
Alanine
Algae
Amino acids
Anions
Carbonyl compounds
Channel gating
Chlorides
Cyanides
E coli
Escherichia coli
Humanities and Social Sciences
Illumination
multidisciplinary
pH effects
Scanning mutagenesis
Science
Science (multidisciplinary)
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep41879