Novel Genetically Encoded Bright Positive Calcium Indicator NCaMP7 Based on the mNeonGreen Fluorescent Protein

Abstract

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 ΔF/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 ΔF/F of 27 and high affinity (Kd 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 Å 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.

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

“…Our group recently published a paper presenting another calcium indicator, named NCaMP7 [9], so we compared the FGCaMP7 indicator to that one. As compared to FGCaMP7, the NCaMP7 indicator [9] belongs to the intensiometric class of GECIs, not ratiometric one.…”

“…Our group recently published a paper presenting another calcium indicator, named NCaMP7 [9], so we compared the FGCaMP7 indicator to that one. As compared to FGCaMP7, the NCaMP7 indicator [9] belongs to the intensiometric class of GECIs, not ratiometric one. NCaMP7 has also a different design with the insertion of CaM/M13-peptide pair into fluorescent protein; FGCaMP7 has M13-like peptide and CaM attached to the N-and C-terminal ends of the circularly permutated EGFP.…”

“…The NTnC-like family preserves the mechanism of fluorescence change common to single FP-based sensors but also possesses advantages such as a reduced number of Ca 2+ -binding sites and small molecular size of the indicator. The ncpGCaMP6s and NCaMP7 indicators contain an insertion of the CaM/M13-peptide pair in EGFP and mNeonGreen proteins, respectively [9,10].…”

“…Changing the topology of the indicator can also reduce the interaction of the GECI with the intracellular environment. FRAP experiments for the NCaMP7 indicator (based on the insertion of the CaM/M13-peptide into mNeonGreen) demonstrated that, when the topology is changed in this way, calcium indicator interactions with the intracellular environment may, to some extent, be prevented [9].…”

Drug Screening with Genetically Encoded Fluorescent Sensors: Today and Tomorrow

“…Though, their field of use is extending to other areas, including for HTS. Using primarily GFP color variants and a series of red and photoconvertible fluorescent proteins in combination with Ca 2+ -sensing domains a wide panel of probes have been developed in recent years [ 118 , 119 , 120 ]. New generation Ca 2+ indicators are characterized by improved sensitivity and dynamic range which make them comparable with widely used standard synthetic dyes.…”