PMID- 32121243 OWN - NLM STAT- MEDLINE DCOM- 20201125 LR - 20201217 IS - 1422-0067 (Electronic) IS - 1422-0067 (Linking) VI - 21 IP - 5 DP - 2020 Feb 28 TI - Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein. LID - 10.3390/ijms21051644 [doi] LID - 1644 AB - Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an DeltaF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an DeltaF/F of 27 and high affinity (K(d) of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 A resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively. FAU - Subach, Oksana M AU - Subach OM AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. FAU - Sotskov, Vladimir P AU - Sotskov VP AUID- ORCID: 0000-0002-1729-5744 AD - Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, Moscow 119991, Russia. FAU - Plusnin, Viktor V AU - Plusnin VV AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. FAU - Gruzdeva, Anna M AU - Gruzdeva AM AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. AD - Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, Moscow 119991, Russia. FAU - Barykina, Natalia V AU - Barykina NV AUID- ORCID: 0000-0001-5835-8388 AD - P.K. Anokhin Research Institute of Normal Physiology, Moscow 125315, Russia. FAU - Ivashkina, Olga I AU - Ivashkina OI AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. AD - Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, Moscow 119991, Russia. AD - P.K. Anokhin Research Institute of Normal Physiology, Moscow 125315, Russia. FAU - Anokhin, Konstantin V AU - Anokhin KV AD - Institute for Advanced Brain Studies, M.V. Lomonosov Moscow State University, Moscow 119991, Russia. AD - P.K. Anokhin Research Institute of Normal Physiology, Moscow 125315, Russia. FAU - Nikolaeva, Alena Y AU - Nikolaeva AY AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. FAU - Korzhenevskiy, Dmitry A AU - Korzhenevskiy DA AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. FAU - Vlaskina, Anna V AU - Vlaskina AV AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. FAU - Lazarenko, Vladimir A AU - Lazarenko VA AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. FAU - Boyko, Konstantin M AU - Boyko KM AD - Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia. FAU - Rakitina, Tatiana V AU - Rakitina TV AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. AD - M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow 117997, Russia. FAU - Varizhuk, Anna M AU - Varizhuk AM AD - Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia. AD - Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Moscow 119435, Russia. FAU - Pozmogova, Galina E AU - Pozmogova GE AD - Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia. AD - Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Moscow 119435, Russia. FAU - Podgorny, Oleg V AU - Podgorny OV AD - M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow 117997, Russia. AD - Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Moscow 117997, Russia. AD - N.K. Koltzov Institute of Developmental Biology, RAS, Moscow 119334, Russia. FAU - Piatkevich, Kiryl D AU - Piatkevich KD AUID- ORCID: 0000-0002-7777-9468 AD - Massachusetts Institute of Technology, Cambridge, MA 02139, USA. AD - Westlake University, Hangzhou, Zhejiang 310024, China. FAU - Boyden, Edward S AU - Boyden ES AD - Massachusetts Institute of Technology, Cambridge, MA 02139, USA. FAU - Subach, Fedor V AU - Subach FV AD - National Research Center "Kurchatov Institute", Moscow 123182, Russia. 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18-34-00640/capital ER, Cyrillicsmall o, Cyrillicsmall es, Cyrillicsmall es, Cyrillicsmall i, Cyrillicsmall short i, Cyrillicsmall es, Cyrillicsmall ka, Cyrillicsmall i, Cyrillicsmall short i, Cyrillic capital EF, Cyrillicsmall o, Cyrillicsmall en, Cyrillicsmall de, Cyrillic capital EF, Cyrillicsmall u, Cyrillicsmall en, Cyrillicsmall de, Cyrillicsmall a, Cyrillicsmall em, Cyrillicsmall ie, Cyrillicsmall en, Cyrillicsmall te, Cyrillicsmall a, Cyrillicsmall el, Cyrillicsmall soft sign, Cyrillicsmall en, Cyrillicsmall yeru, Cyrillicsmall ha, Cyrillic capital I, Cyrillicsmall es, Cyrillicsmall es, Cyrillicsmall el, Cyrillicsmall ie, Cyrillicsmall de, Cyrillicsmall o, Cyrillicsmall ve, Cyrillicsmall a, Cyrillicsmall en, Cyrillicsmall i, Cyrillicsmall short i, Cyrillic (capital ER, Cyrilliccapital EF, Cyrilliccapital EF, Cyrilliccapital I, Cyrillic)/ GR - 19-34-70004/capital ER, Cyrillicsmall o, Cyrillicsmall es, Cyrillicsmall es, Cyrillicsmall i, Cyrillicsmall short i, Cyrillicsmall es, Cyrillicsmall ka, Cyrillicsmall i, Cyrillicsmall short i, Cyrillic capital EF, Cyrillicsmall o, Cyrillicsmall en, Cyrillicsmall de, Cyrillic capital EF, Cyrillicsmall u, Cyrillicsmall en, Cyrillicsmall de, Cyrillicsmall a, Cyrillicsmall em, Cyrillicsmall ie, Cyrillicsmall en, Cyrillicsmall te, Cyrillicsmall a, Cyrillicsmall el, Cyrillicsmall soft sign, Cyrillicsmall en, Cyrillicsmall yeru, Cyrillicsmall ha, Cyrillic capital I, Cyrillicsmall es, Cyrillicsmall es, Cyrillicsmall el, Cyrillicsmall ie, Cyrillicsmall de, Cyrillicsmall o, Cyrillicsmall ve, Cyrillicsmall a, Cyrillicsmall en, Cyrillicsmall i, Cyrillicsmall short i, Cyrillic (capital ER, Cyrilliccapital EF, Cyrilliccapital EF, Cyrilliccapital I, Cyrillic)/ GR - 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Journal Article DEP - 20200228 PL - Switzerland TA - Int J Mol Sci JT - International journal of molecular sciences JID - 101092791 RN - 0 (Indicators and Reagents) RN - 147336-22-9 (Green Fluorescent Proteins) RN - SY7Q814VUP (Calcium) SB - IM MH - Animals MH - Behavior, Animal MH - Calcium/*metabolism MH - Cells, Cultured MH - Crystallography, X-Ray MH - Fluorometry MH - *Genetic Techniques MH - Green Fluorescent Proteins/*metabolism MH - HeLa Cells MH - Hippocampus/metabolism MH - Humans MH - Indicators and Reagents MH - Kinetics MH - Mice, Inbred C57BL MH - Models, Molecular MH - Neurons/metabolism MH - Photons MH - Visual Cortex/physiology MH - Wakefulness PMC - PMC7084697 OTO - NOTNLM OT - NCaMP7 OT - calcium imaging OT - crystal structure OT - fluorescent protein OT - genetically encoded calcium indicator (GECI) OT - high brightness OT - protein engineering COIS- The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. EDAT- 2020/03/04 06:00 MHDA- 2020/11/26 06:00 PMCR- 2020/03/01 CRDT- 2020/03/04 06:00 PHST- 2020/02/08 00:00 [received] PHST- 2020/02/25 00:00 [revised] PHST- 2020/02/25 00:00 [accepted] PHST- 2020/03/04 06:00 [entrez] PHST- 2020/03/04 06:00 [pubmed] PHST- 2020/11/26 06:00 [medline] PHST- 2020/03/01 00:00 [pmc-release] AID - ijms21051644 [pii] AID - ijms-21-01644 [pii] AID - 10.3390/ijms21051644 [doi] PST - epublish SO - Int J Mol Sci. 2020 Feb 28;21(5):1644. doi: 10.3390/ijms21051644.