A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites

Barykina, N. V., Subach, O. M., Doronin, D. A., Sotskov, V. P., Roshchina, M. A., Kunitsyna, T. A., Malyshev, A. Y., Smirnov, I. V., Azieva, A. M., Sokolov, I. S., Piatkevich, K. D., Burtsev, M. S., Varizhuk, A. M., Pozmogova, G. E., Anokhin, K. V., Subach, F. V., Enikolopov, G. N. (2016) A new design for a green calcium indicator with a smaller size and a reduced number of calcium-binding sites. Sci Rep, 6. p. 34447. ISSN 2045-2322 (Electronic)2045-2322 (Linking)

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URL: https://www.ncbi.nlm.nih.gov/pubmed/27677952
DOI: 10.1038/srep34447

Abstract

Genetically encoded calcium indicators (GECIs) are mainly represented by two- or one-fluorophore-based sensors. One type of two-fluorophore-based sensor, carrying Opsanus troponin C (TnC) as the Ca2+-binding moiety, has two binding sites for calcium ions, providing a linear response to calcium ions. One-fluorophore-based sensors have four Ca2+-binding sites but are better suited for in vivo experiments. Herein, we describe a novel design for a one-fluorophore-based GECI with two Ca2+-binding sites. The engineered sensor, called NTnC, uses TnC as the Ca2+-binding moiety, inserted in the mNeonGreen fluorescent protein. Monomeric NTnC has higher brightness and pH-stability in vitro compared with the standard GECI GCaMP6s. In addition, NTnC shows an inverted fluorescence response to Ca2+. Using NTnC, we have visualized Ca2+ dynamics during spontaneous activity of neuronal cultures as confirmed by control NTnC and its mutant, in which the affinity to Ca2+ is eliminated. Using whole-cell patch clamp, we have demonstrated that NTnC dynamics in neurons are similar to those of GCaMP6s and allow robust detection of single action potentials. Finally, we have used NTnC to visualize Ca2+ neuronal activity in vivo in the V1 cortical area in awake and freely moving mice using two-photon microscopy or an nVista miniaturized microscope.

Item Type: Paper
Subjects: Investigative techniques and equipment > microscopy
Investigative techniques and equipment > optical devices > microscope techniques or equipment
CSHL Authors:
Communities: CSHL labs > Enikopolov lab
Depositing User: Matt Covey
Date: 2016
Date Deposited: 07 Oct 2016 18:59
Last Modified: 09 Nov 2017 21:33
PMCID: PMC5039633
Related URLs:
URI: http://repository.cshl.edu/id/eprint/33627

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