Improved Orange and Red Ca2+ Indicators and Photophysical Considerations for Optogenetic Applications

被引：175

作者：

Wu, Jiahui ^{[1
]}

Liu, Lin ^{[2
,3
]}

Matsuda, Tomoki ^{[4
]}

Zhao, Yongxin ^{[1
]}

Rebane, Aleksander ^{[5
]}

Drobizhev, Mikhail ^{[5
]}

Chang, Yu-Fen ^{[4
]}

Araki, Satoko ^{[4
]}

Arai, Yoshiyuki ^{[4
]}

March, Kelsey ^{[6
]}

Hughes, Thomas E. ^{[6
]}

Sagou, Ken ^{[7
]}

Miyata, Takaki ^{[7
]}

Nagai, Takeharu ^{[4
]}

Li, Wen-hong ^{[2
,3
]}

Campbell, Robert E. ^{[1
]}

机构：

[1] Univ Alberta, Dept Chem, Edmonton, AB T6G 2G2, Canada

[2] Univ Texas SW Med Ctr Dallas, Dept Cell Biol, Dallas, TX 75390 USA

[3] Univ Texas SW Med Ctr Dallas, Dept Biochem, Dallas, TX 75390 USA

[4] Osaka Univ, Inst Sci & Ind Res, Osaka 5670047, Japan

[5] Montana State Univ, Dept Phys, Bozeman, MT 59717 USA

[6] Montana State Univ, Dept Cell Biol & Neurosci, Bozeman, MT 59717 USA

[7] Nagoya Univ, Grad Sch Med, Showa Ku, Nagoya, Aichi 4668550, Japan

来源：

ACS CHEMICAL NEUROSCIENCE | 2013年 / 4卷 / 06期

基金：

加拿大自然科学与工程研究理事会;

关键词：

Ca2+ imaging; fluorescent proteins; Ca2+ indicators; channelrhodopsin; photoactivation; FLUORESCENT PROTEIN; STRUCTURAL BASIS; EXCITATION; CHANNELRHODOPSINS; VARIANTS; COLOR;

D O I：

10.1021/cn400012b

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

We have used protein engineering to expand the palette of genetically encoded calcium ion (Ca2+) indicators to include orange and improved red fluorescent variants, and validated the latter for combined use with optogenetic activation by channelrhodopsin-2 (ChR2). These indicators feature intensiometric signal changes that are 1.7- to 9.7-fold improved relatively to the progenitor Ca2+ indicator, R-GEC01. In the course of this work, we discovered a photoactivation phenomenon in red fluorescent Ca2+ indicators that, if not appreciated and accounted for, can cause false-positive artifacts in Ca2+ imaging traces during optogenetic activation with ChR2. We demonstrate, in both a beta cell line and slice culture of developing mouse neocortex, that these artifacts can be avoided by using an appropriately low intensity of blue light for ChR2 activation.

引用

页码：963 / 972

页数：10

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