期刊
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
卷 21, 期 18, 页码 -出版社
MDPI
DOI: 10.3390/ijms21186883
关键词
genetically encoded calcium indicator; protein engineering; calcium imaging; bacterial phytochrome; GAF-CaMP3; GECI; near-infrared; fluorescent protein
资金
- National Research Center Kurchatov Institute [1360]
- RSF [16-15-10323]
- RFBR [19-04-00395]
- Russian Science Foundation [16-15-10323] Funding Source: Russian Science Foundation
The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2-superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2-sfGFP indicator, named GAF-CaMP3-sfGFP. The GAF-CaMP3-sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger Delta F/F response to calcium ions. As compared to GAF-CaMP2-sfGFP, in cultured HeLa cells, GAF-CaMP3-sfGFP had similar brightness but a 1.9-fold larger Delta F/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3-sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3-sfGFP showed a linear Delta F/F response in the range of 0-20 APs and in this range demonstrated a 1.4-fold larger Delta F/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.
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