First we compared the break points of circular permutation within the β-barrel of fluorescent proteins used in Pericams and GCaMPs Ca²⁺ sensors (Table 1A). Remarkably, amino acid residues corresponding to the positions 145 and 148 of the intact Aequorea victoria GFP (avGFP) (Figure 1a) were shown to substantially determine the fluorescent properties of the sensors 78. Therefore, it is likely that in Pericams and GCaMPs the amino acid residues 148 and 145 are in a close proximity to the chromophore, similarly to the native avGFP (PDB ID: 1GFL) 15. The amino acid residue preceding position 148 and the one following position 145 should be the outermost within the cpFP beta-barrel (Figure 1b). It can be presumed that the positional relationship of these key amino acid residues and the sensitive domains is common for the sensors described in Refs. 78101112 and this family of circularly permuted variants can be generally named cpFP147-146. A similar spatial organization can be proposed for the Camgaroo sensors 69.

Looking for the sensor variants with expanded dynamic range, we generated a set of Ca²⁺ sensor constructs which varied at positions 148 and 145. In GCaMPs and Pericams position 145 was either Gly or Thr (Table 1A). We presumed that two other amino acid residues with rather compact side chains, Ser and Ala, wouldn't cause spatial conflict at position 145, while they would alter the spectral properties of the sensor. Using site-directed mutagenesis, we ntroduced either Ser, Ala or Thr in position 145, in combination with Asp148 (which earlier resulted in Ratiometric Pericam), Glu148 (used in GCaMPs), or Asn148. The overall sensors design was similar to that of GCaMPs. We used M13 peptide, a calmodulin domain and linkers lengths identical to those reported for GCaMP1 8, combined with cpFP described in Ref. 10 and originating from Ratiometric Pericam 7. Importantly, all our sensor variants carried Phe at position 203, which was earlier shown to influence the fluorescent properties of cpFP-based indicators 7.

Our sensor variants and their responses to Ca²⁺ are summarized in Table 1B. Indeed, the variation at positions 145 and 148 was shown to determine sensor characteristics to a large extent. Analogously to Ratiometric Pericam (carrying Gly145), combination of Asp148 and Phe203 resulted in dual-excitation spectrum (green fluorescence excited at 400 nm and 490 nm) and strong ratiometric response to Ca²⁺ when coupled with Ala145 or Ser145. However, combination Asp148/Phe203/Thr145 resulted in poorly responding sensor.

At the same time, all variants carrying Asn148 or Glu148 showed a single 490 nm excitation peak. While Asn148-containing sensors demonstrated low dynamic ranges, combinations Glu148/Thr145 (cps2) and Glu148/Ser145 (cps2(S)) resulted in high dynamic range sensors with up to 14.5-fold increase of 490 nm excited green fluorescence in response to 1 mM Ca²⁺. Further, cps2 was optimized in respect of folding at 37°C using random mutagenesis approach, resulting in an enhanced variant with 12-fold fluorescence increase in response to Ca²⁺, named Case12. This variant was characterized by a fast maturation and high brightness and was chosen for further tests in living cells. Site directed mutagenesis T145S of Case12 resulted in a sensor named Case16, which was characterized by the highest dynamic range, with up to 16.5-fold fluorescence increase in response to Ca²⁺ (Table 1C), though less effective maturation in E. coli.

Spectral properties of Case12 and Case16 are summarized in Figure 2 and Table 2. Both sensors demonstrated record multi-fold increase of green fluorescent signal in response to 1 mM Ca²⁺ (Figure 2a,b). Changes in the absorbance spectra in response to Ca²⁺ (Figure 2c,d) were quite predictable from the observed fluorescence changes and reflected the redistribution between neutral and charged states of the chromophore in favor of the latter one. Similar responses, although with lower dynamic ranges, were reported earlier for Flash-pericam 7 and Camgaroo 6 sensors. Importantly, maximum brightness (in the presence of 1 mM Ca²⁺, at pH 7.4) reached 0.35 and 0.28 of that of EGFP for Case12 and Case16, respectively, which is essentially higher than that of Flash-pericam or GCaMP1.6 and comparable to GCaMP2 (Table 2).

The common weak point of cpFP-based sensors is their low pH stability. For example, pKa (value of pH at which fluorescence brightness is 50% of maximum) for Pericams ranges from 7.9 to 8.5. Therefore, at physiological ranges of pH (7.2–7.5) such sensors exhibit lower brightness and dynamic range 7. In contrast, the pKa values of Case12 and Case16 were shown to be 7.2 (in the presence of 0.2 mM Ca²⁺) close to that reported for GCaMP1 (Table 2 and Figure 2e,f). Therefore, Case12 and Case16 are characterized by notably higher pH-stability compared to GCaMP1.6 or Pericams (Table 2).

Further we measured Case12 and Case16 K_d values for Ca²⁺ binding at pH 7.4, using Molecular Probes assay (Calcium Calibration Buffer Kit #1). The apparent K_d was found to be 1 μM for both sensors (Figure 2g,h), which lies within the physiological range of Ca²⁺ concentrations. In most eukaryotic cells the free Ca²⁺ concentration rests at approximately 100–200 nM and following activation of cellular signaling pathways cytosolic Ca²⁺ levels are elevated up to several μM 16171819.

First, we checked maximum fluorescence response for both sensors in living HeLa cells. For this purpose the sensors were cloned into the N-vector (Evrogen), driving gene expression in eukaryotic cells. Cells transfected with Case12 sensor variant demonstrated relatively weak green fluorescence, which could be detected under a FITC filter set or upon excitation with a 488 nm laser line, emission collected at 500–540 nm (Leica microscope DM IRE2, confocal TCS-SP2, objective HCX-PL-APO-63x/1.40-0.60/OIL). Addition of 30 μM Ca²⁺ ionophore A23187, allowing Ca²⁺ (2 mM Ca²⁺ in the medium) to enter cells, typically resulted in 5–6-fold increase in green fluorescence brightness. Further addition of 20 mM EGTA removed Ca²⁺ and decreased the fluorescence signal close to baseline level, with the final contrast of 11–12-fold. Very similar results with 7–9 fold increase in the presence of ionophore and the final contrast of 13–15 fold were obtained for Case16 (Figure 3a–d). In analogues experiments with GCaMP2, one of the best reported cpFP-based Ca²⁺ sensors 12 (a kind gift from Prof. Michael I. Kotlikoff and Dr. Junichi Nakai), the corresponding contrasts were 2.5-fold increase in the presence of ionophore and 5-fold decrease after addition of EGTA, corresponding with the reported dynamic range 12.

Further we tested the response of the sensors to the less pronounced Ca²⁺ changes under physiological conditions. M21 human melanoma cells expressing Case12 displayed a nice high dynamic range response to ATP at a final concentration of 100 μM (Figure 3e–h). This experiment clearly showed that Case12 fluorescence response to Ca²⁺ oscillations is fast and reversible. It also demonstrated that the sensor responds to changes in Ca²⁺ concentration in living cells in the nanomolar range.

In another experimental set-up, we monitored Ca²⁺ responses in PC12 cells. Transfected cells were grown at 37°C and NGF (nerve growth factor) was added 24 hours before the experiment in order to induce differentiation. In the course of imaging, cells were perfused with carbogen-saturated ACSF (artificial cerebro-spinal fluid – modified Ringer) 20 within a 34°C chamber. Figure 3i shows the Ca²⁺ response of Case12 sensor to carbachol (at concentration 500 μM, between 170 and 350 s). After 10 min of wash, 30 mM KCl was added to the same cells (Figure 3j).

Further we compared Ca²⁺ responses of Case12, Case16 and a well-known chemical probe for Ca²⁺, Oregon Green 488 BAPTA-1 (Molecular Probes; loaded at a final concentration of 7 μM for 4 hours) to 30 mM KCl in PC12 cells. Initial fluorescence brightness of the genetically encoded sensors and Oregon Green were approximately at the same level, and amplitudes of responses to Ca²⁺ were very similar. This indicated that Case12 and Case16 are comparable with chemical probes for Ca²⁺ in respect of signal brightness and dynamic range.

To further evaluate the sensors in comparison with chemical probes, we applied Case12 for measuring the Ca²⁺ response to a prolonged glutamate treatment (100 μM glutamate, 10 μM glycine) in cortical neurons. Figure 4 illustrates typical response of cortical neuron transfected with Case12 and loaded with fura-2FF, which is a widely used chemical Ca²⁺ indicator. Clear spectral differences allowed monitoring of fluorescence of both Case12 and fura-2FF sensors independently, in sequential excitation mode. While fura-2FF response was tracked by 340 nm and 380 nm excited green fluorescence brightness (505–530 nm emission filter), Case12 fluorescence was monitored using 515–565 nm emission filter, with excitation at 490 nm. Signals were measured within the same cells in parallel (Figure 4). Stimulation of glutamate receptors induced primary increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) that after a certain delay was superseded by the secondary [Ca²⁺]_c elevation referred to as loss of Ca²⁺ homeostasis 2122. The signal of Case12 clearly detected both phases of glutamate-induced [Ca²⁺]_c elevation (Figure 4). It is fair to note that the latent period between these phases was seen as a pronounced decrease in the Case12 fluorescence, while the fura-2FF ratio showed increased [Ca²⁺]_c level during this period. Taking into account that cpFP-based sensors are sensitive to pH changes, we presume that this decrease in Case12 signal is caused by glutamate-induced decrease in cytosolic pH 212324. Glutamate washout by Ca²⁺-free EGTA (100 μM)-containing solution induced delayed [Ca²⁺]_c recovery. Consequent application of protonophore FCCP (1 μM) in Ca²⁺-free medium caused mitochondrial depolarization and released Ca²⁺ accumulated by mitochondria during glutamate treatment 2526. Both processes were clearly monitored by the Case12 signal (Figure 4). In spite of the dramatic pH changes during glutamate treatment of cortical neurons, Case12 clearly monitored all the changes in ([Ca²⁺]_c).

Sensor constructs generation and site directed mutagenesis were performed by overlap-extension PCR 28, with primers containing the appropriate target substitutions. The cDNA coding the circularly permuted green fluorescent protein was amplified from the HyPer sensor 10. The sequence of the M13 peptide (from the chicken smooth muscle myosine light chain kinase) was generated by step-out PCR. The sequence of calmodulin was amplified from a human cDNA library. Clontech Diversity PCR Random Mutagenesis kit was used for random mutagenesis, in conditions optimal for 7 mutations per 1000 bp. For bacterial expression, a PCR-amplified BamHI/HindIII fragment encoding sensor variant was cloned into the pQE30 vector (Qiagen). For expression in eukaryotic cells, PCR-amplified AgeI/NotI fragment encoding the corresponding sensor variant was swapped for TurboGFP within the pTurboGFP-N vector (Evrogen).

Proteins fused to the N-terminal polyhistidine tag were expressed in E. coli XL1 Blue strain (Invitrogen). The bacterial cultures were centrifuged and the cell pellets re-suspended in 20 mM Tris-HCl, 100 mM NaCl, pH 7.4 buffer and lysed by sonication. The recombinant proteins were purified using TALON metal-affinity resin (Clontech) followed by a desalting step over gel-filtration columns (Bio-Rad). Absorption spectra were recorded with a Beckman DU520 UV/VIS Spectrophotometer. A Varian Cary Eclipse Fluorescence Spectrophotometer was used for measuring excitation-emission spectra. For calculation of the molar extinction coefficients, we relied on the determination of the mature chromophore concentration. Proteins were alkali-denatured with an equal volume of 2 M NaOH. Under these conditions, the GFP chromophore absorbs at 446 nm and its molar extinction coefficient equals 44,000 M^-1cm^-1. Absorption spectra for native and alkali-denatured proteins were analyzed. Based on the absorption of denatured proteins, molar extinction coefficients for the native states were estimated. For determination of the quantum yield, the fluorescence of the mutants was compared with equally absorbing EGFP (quantum yield 0.60 29). Ca²⁺ titrations were performed using buffers identical to Calcium Calibration Buffer Kit #1 (Molecular Probes), yielding the free Ca²⁺ concentrations from zero to 39 μM. Fluorescence emission spectra (excited at 490 nm) were recorded at 22°C. pH titrations were performed by using a series of buffers. For each pH value an aliquot of purified protein was diluted in an equal volume of the corresponding buffer solution either in the presence of 5 mM EGTA or 200 μM CaCl₂, and the fluorescence brightness was measured (fluorescence excited at 490 nm and detected at 520 nm).

M21 human melanoma cells were transfected with FuGENE reagent (Roche), grown for 48 h in RPMI medium in the presence of 10% FBS (Invitrogen) in a CO₂ incubator at 37°C, 5% CO2, and then incubated for 1 h before imaging in 10% FBS Ham's F-12 medium (Invitrogen). Imaging was then performed in Ham' s F-12 medium by confocal laser scanning microscopy (LSM510, Carl Zeiss Microimaging, Inc.) ATP was added to a final concentration of 100 μM and the fluorescence of selected transfected cells was then monitored every 0.294 sec. Data were analyzed using ImageJ software (NIH, Bethesda).

Cortical neurons were prepared from 1–3 days old newborn Wistar rats. Cortical tissue was minced in ice-cold Krebs salt solution. (mM: 130 NaCl, 5.4 KCl, 20 HEPES, 0.4 KH₂PO₄, 15 Glucose, 0.5 MgSO₄ and 3 mg/ml BSA (Sigma), pH 7.4), then the tissue was digested in Krebs solution with 0.8 mg/ml trypsin 1–300 (ICN) for 15 min at 36°C. Trypsin was inactivated by washing with Krebs solution containing 0.08 mg/ml trypsin inhibitor (Sigma) and 0.01 mg/ml DNase (Roche). Cells were dissociated by trituration and pelleted in Krebs solution containing 0.5 mg/ml trypsin inhibitor and 0.08 mg/ml DNase. Cells were then resuspended in Neurobasal Medium (Gibco) with supplement B-27 (Gibco), GlutaMax (Gibco) and penicilline/streptomycine (Gibco) and plated onto 25 mm glass coverslips, coated with poly-D-lysine (Sigma). After 5–7 days in the primary culture, cells were transfected with Case12 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the protocol recommended by the manufacturer. Fluorescence analyses were carried out 2 d after transfection.

The neurons transfected with Case12 were loaded for 40 min with 3 μM fura-2FF/AM (Teflabs, Austin, TX, USA) in the incubator in the presence of cell culture medium. Images were acquired on an epifluorescence inverted microscope Axiovert 200 (Zeiss, Germany) equipped with a 20× fluorite objective. [Ca²⁺]_c was monitored in single cells using excitation light provided by a Xenon arc lamp, the beam passing sequentially through 10 nm bandpass filters centered at 340, 380 and 490 nm housed in a computer-controlled filter wheel (Sutter Instrument Co., CA, USA). Emitted fluorescence light was reflected through a 505–530 nm filter (fura-2FF) and 515–565 nm filter (Case12) placed in computer-controlled filter wheel. Images were acquired by CCD camera (Roper Scientific, USA). All imaging data were collected and analyzed using the Metafluor 6.1 software (Universal Imaging Corp., USA). The fura-2FF data are presented as the ratio of light excited at 340 nm/380 nm.

The coverslips with cell culture were placed into the 300 μl experimental chamber at room temperature (25°C) and washed with a standard physiological recording saline containing (mM): 140 NaCl, 5.4 KCl, 2 MgCl₂, 2 CaCl₂, 5 glucose and 20 HEPES, pH adjusted to 7.4 with NaOH. The solution in the chamber was removed by a peristaltic pump. The solutions were added into the chamber with the aid of a pipette. Washout of solutions was made three times to completely remove old solution from the chamber.