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

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.

A bright and high-performance genetically encoded Ca2+indicator based on mNeonGreen fluorescent protein

“…In the Ca 2+ bound state, mNG-GECO1 has an extinction coefficient of 102,000 M −1 cm −1 and quantum yield of 0.69, giving it an overall brightness (= EC * QY) of 70. This value is similar to the value of 77 previously reported for NTnC 18 and 78% of the brightness of mNG itself (measured by us to be 112,000 M −1 cm −1 * 0.8 = 90) ( Supplementary Fig. 4 ).…”

“…However, reduced expression requires increased intensity of excitation light to achieve an equivalent fluorescent signal, which can lead to increased phototoxicity and photobleaching. Another solution is to reduce the number of Ca 2+ binding sites like that in the TnC-based GECIs, NTnC 18 and YTnC 19 . Unfortunately, these indicators have relatively low fluorescence response (ΔF/F min ~ 1 for NTnC and ~ 10.6 for YTnC) compared to the recent GCaMP7 variants (ΔF/F min ~ 21 to 145) 20 .…”

Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi

“…Ca 2+ (varying concentrations) and protein solutions (20 μg/ml in 30 mM HEPES buffer (pH 7.2) containing 100 mM KCl) were prepared as described in [5]. Fluorescence excitation was set to 500 nm for GCaMP6s and to 500 or 402 nm for FGCaMP.…”

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

“…It is well known that intracellular acidification of 0.3–0.4 pH units is a common feature of apoptotic cell death [39, 40]. Calcium-bound GCaMP6s fluorescence is stable from pH 6.5–9.5 [41], suggesting that GCaMP6s performance would not be appreciably affecting by pH during cell death. Furthermore, our data does not indicate any significant loss of GCaMP6s fluorescence over the time courses being investigated in our model system.…”