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 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.
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Satoko
S
Doi
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Takashi
T
Tsukamoto
Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277-8564 Japan.
Susumu
S
Yoshizawa
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan.
Yuki
Y
Sudo
D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000737:chemistry; D000075402:Channelrhodopsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D055672:Static Electricity; D014466:Ultraviolet Rays
D000075402:Channelrhodopsins; D002712:Chlorides; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D001120:Arginine