TY - JOUR
T1 - Green-sensitive, long-lived, step-functional anion channelrhodopsin-2 variant as a high-potential neural silencing tool
AU - Kojima, Keiichi
AU - Miyoshi, Natsuki
AU - Shibukawa, Atsushi
AU - Chowdhury, Srikanta
AU - Tsujimura, Masaki
AU - Noji, Tomoyasu
AU - Ishikita, Hiroshi
AU - Yamanaka, Akihiro
AU - Sudo, Yuki
N1 - Funding Information:
We thank Dr. Hiromu Yawo for technical support for electrophysiological experiments. We also thank Ms. Satoko Doi for the preparation of purified GtACR2. This work was financially supported by JSPS KAKENHI grant numbers JP19K16090 to K.K., JP17K18013 to T.N., JP18H05155, JP18H01937, JP20H03217, and JP20H05090 to H.I., and JP18H02411, JP19H04727, and JP19H05396 to Y.S. This research was partially supported by JST CREST (JPMJCR1656) to H.I., A.Y., and Y.S. and AMED (20dm0207060h0004) to Y.S.
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/8/6
Y1 - 2020/8/6
N2 - Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the Escherichia coli expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λmax = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2"from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.
AB - Anion channelrhodopsin-2 (GtACR2) was identified from the alga Guillardia theta as a light-gated anion channel, providing a powerful neural silencing tool for optogenetics. To expand its molecular properties, we produced here GtACR2 variants by strategic mutations on the four residues around the retinal chromophore (i.e., R129, G152, P204, and C233). After the screening with the Escherichia coli expression system, we estimated spectral sensitivities and the anion channeling function by using the HEK293 expression system. Among the mutants, triple (R129M/G152S/C233A) and quadruple (R129M/G152S/P204T/C233A) mutants showed the significantly red-shifted absorption maxima (λmax = 498 and 514 nm, respectively) and the long-lived channel-conducting states (the half-life times were 3.4 and 5.4 s, respectively). In addition, both mutants can be activated and inactivated by different wavelengths, representing their step-functional ability. We nicknamed the quadruple mutant "GLaS-ACR2"from its green-sensitive, long-lived, step-functional properties. The unique characteristics of GLaS-ACR2 suggest its high potential as a neural silencing tool.
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U2 - 10.1021/acs.jpclett.0c01406
DO - 10.1021/acs.jpclett.0c01406
M3 - Article
C2 - 32697087
AN - SCOPUS:85089173925
SN - 1948-7185
VL - 11
SP - 6214
EP - 6218
JO - Journal of Physical Chemistry Letters
JF - Journal of Physical Chemistry Letters
IS - 15
ER -