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

Natalia V Barykina,Anna M Varizhuk,Aleksey Y Malyshev,Danila A Doronin,Ilya S Sokolov,Konstantin V Anokhin,Vladimir P Sotskov,Tatiana A Kunitsyna,Fedor V Subach,Marina A Roshchina,Oksana M Subach,Ivan V Smirnov,Mikhail S Burtsev,Asya M Azieva,Grigori N Enikolopov,Galina E Pozmogova,Kiryl D Piatkevich

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.

Highlights

Size-exclusion chromatography was performed with a SuperdexTM 200 10/300 GL column using GE ÄKTA Explorer (Amersham Pharmacia, UK) FPLC System
Using whole-cell patch clamp recording, we have revealed similar kinetics of Ca2+ responses in neurons expressing NTnC and GCaMP6s
We expect that further exploration of NTnC-like designs, with the aim of enhancing its ΔF/F dynamic range, may result in sensors with performance levels similar or superior to those of GECIs with conventional designs

Summary

Results and Discussion

Development of a novel green fluorescent calcium sensor, NTnC. To design a small sensor with two binding sites for calcium ions, we selected as the sensory part the C-terminal minimal domain of TnC, which is expressed in the swim bladder and white muscles of O. tau (tsTnC). The decreased ΔF/F response of the NTnC calcium sensor observed during spontaneous activity of neuronal cultures, compared with those of pure protein and expression in HeLa cells, could be partially explained by the relatively high concentration of free Ca2+ in resting neurons, which has been estimated as 50–100 nM (increasing 5-fold to 100-fold in active neurons)[16,17]. We have found that NTnC shows sensitivity to single APs similar to that of GCaMP6s This sensitivity correlates with the higher affinity of NTnC to Ca2+ ions and with its linear response at low Ca2+ concentration changes, which is due to the decreased number of Ca2+-binding sites. We expect that further exploration of NTnC-like designs, with the aim of enhancing its ΔF/F dynamic range, may result in sensors with performance levels similar or superior to those of GECIs with conventional designs

Materials and Methods

Author Contributions

Additional Information