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NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium res...

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Detalles Bibliográficos
Autores principales: Barykina, Natalia V., Doronin, Danila A., Subach, Oksana M., Sotskov, Vladimir P., Plusnin, Viktor V., Ivleva, Olga A., Gruzdeva, Anna M., Kunitsyna, Tatiana A., Ivashkina, Olga I., Lazutkin, Alexander A., Malyshev, Aleksey Y., Smirnov, Ivan V., Varizhuk, Anna M., Pozmogova, Galina E., Piatkevich, Kiryl D., Anokhin, Konstantin V., Enikolopov, Grigori, Subach, Fedor V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189086/
https://www.ncbi.nlm.nih.gov/pubmed/30323302
http://dx.doi.org/10.1038/s41598-018-33613-6
Descripción
Sumario:The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.