Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics

D A Doronin,O A Ivleva,V P Sotskov,F V Subach,V V Plusnin,K D Piatkevich,G E Pozmogova,N V Barykina,A M Varizhuk,O M Subach,G N Enikolopov,I V Smirnov,E A Isaakova,A Y Malyshev,K V Anokhin

doi:10.1186/s12896-018-0417-2

Abstract

BackgroundThe recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC’s high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics.ResultsHerein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope.ConclusionsWe demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

Highlights

The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein
We chose the EYFP protein because it has been previously used in GECI with a high fluorescence contrast, such as Camgaroo1 [8]
After the second step of screening, we found that clones from the L1 library exhibited both direct and inverted fluorescence responses with similar fluorescence contrasts of 2.8- and 2.5-fold which corresponded to 1.8- and 1.5fold ΔF/F increase to NTnC, respectively

Summary

Introduction

The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC’s high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+/F+Ca2+) and slow Ca2+ dissociation kinetics. The advantage of the Twitch FRET indicators over the majority of other GECIs is the minimal number of calcium ion-binding sites per molecule that ensures less influence on the cellular concentration of free calcium ions [3]. While FRET GECIs are sensitive and specific to Ca2+ ions, their application is limited, in particular, because of expensive equipment required for their imaging, low fluorescence contrast, and their large molecular size

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Conclusion