Single fluorescent protein-based Ca2+sensors with increased dynamic range
© Souslova et al; licensee BioMed Central Ltd. 2007
Received: 19 February 2007
Accepted: 29 June 2007
Published: 29 June 2007
Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).
Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons.
We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.
An increasing number of genetically encoded fluorescent sensors have recently been developed on the basis of GFP-like proteins [1–3]. However, currently available genetically encoded sensors are characterized by low signal intensity and limited dynamic range (maximum change in fluorescence ratio or intensity) [1, 4, 5], insufficient for routine applications in high throughput screening (HTS) assays and restricting sensitivity of precise single-cell studies. At the same time, genetically encoded sensors provide a much wider flexibility, allowing to be targeted to any chosen cellular compartment, to generate stable cell lines and transgenic animals, to be expressed in a particular tissue and/or in a temporally controlled manner under a specific promoter. Therefore, development of genetically encoded sensors characterized by increased dynamic range and signal intensity remains an actual task.
One of the most promising approaches to create genetically encoded sensors is based on the circularly permuted fluorescent protein (cpFP) fused to or inserted into sensitive domain(s) [6–12]. In the presence of an analyte or in response to a cellular event, sensitive domain(s) undergoe(s) structural rearrangements, inducing conformational changes of cpFP and resulting in its altered fluorescent properties. Circular permutation allows placing sensitive domains in a close proximity to the chromophore environment of cpFP within chimeric sensor construct. Therefore, conformational changes of the sensitive domains and their influence on the spectral properties of cpFP is direct and can lead to significant changes in the fluorescent signal.
In particular, such Ca2+ sensors as GCaMPs [8, 12] and Pericams  were constructed by fusing calmodulin and its target peptide M13 (fragment of myosin light chain kinase) to cpFP. In the presence of Ca2+, calmodulin binds to the M13 peptide, causing conformational changes in the vicinity of the chromophore and thereby influencing cpFP fluorescence. Similar sensors, named Camgaroos [6, 9], are formally based on the non-permuted GFP, but contain an inserted calmodulin molecule at position Tyr145 of EYFP, which is essentially similar to the circular permutation approach.
In most cases, it was shown that spectral changes of the cpFP-based sensors fluorescence occur through the chromophore transition from the neutral (protonated) to the charged (anionic) form. Noteworthy, the same mechanism leads to 100–400 fold increase of green fluorescence after photoactivation of so called photoactivatable fluorescent proteins, PA-GFP  and PS-CFP . This indicates that potentially the same dynamic ranges may be achieved for the cpFP-based fluorescent sensors, provided that the amino acid residues surrounding the chromophore are adapted properly and the conformational changes of the sensitive domains cause favorable conformational alterations in cpFP.
Here we describe the development of high dynamic range cpFP-based Ca2+ sensors, that show up to 16.5-fold increase of the fluorescent signal (F/F0, fluorescence increase, fold) in response to Ca2+. These sensors are more pH stable compared to Flash-pericam  and GCaMP1.6  and have approximately 3-fold higher dynamic range compared to GCaMPs [8, 12]. We believe that the "fluorescent core" of the sensors reported herein may be employed to develop sensors of various specificity with increased dynamic range, allowing reliable quantitative analyses of cellular signaling pathways.
Results and discussion
1. Analyzing the break point
Key amino acid positions, linkers and in vitro Ca2+ response of cpFP-based Ca2+ sensors. Linker sites: linkers connecting M13 with cpFP and cpFP with calmodulin. cpFP is underlined, positions 148 and 145 are shown in bold. Data for Part A are from literature. See Methods section for the details concerning proteins purification and spectroscopy for Parts B and C
Maximum fluorescence response to Ca2+, fold, comments
Part A: previously developed sensors
YNS H NVY---LEYN G TGDQ
3,0 × increase
YNS H NVY---LEYN G TGDQ
8,0 × increase low pH stability
YNS D NVY---LEYN G TDQL
10,0 × ratiometric changes
YNS T NVY---LEYN G TDQL
5,0 × decrease
SSL E NVY---LEYN G TRDQ
4,3 × increase
SSL E NVY---LEYN T RDQL
4,3 × increase brighter than G18 due to 145T
SSL E NVY---LEYN T RDQL
4.9 × increase much brighter than GCaMP1
SSL E NVY---LEYN T RDQL
5 × increase much brighter than GCaMP1.6
Part B: our sensors developed as a result of 145/148 positions mutagenesis
SSL N NVY---LEYN T RDQL
SSL N NVY---LEYN A RDQL
1,4 × decrease
SSL N NVY---LEYN S RDQL
1,5 × increase
SSL E NVY---LEYN T RDQL
14,5 × increase
SSL E NVY---LEYN A RDQL
SSL E NVY---LEYN S RDQL
14,5 × increase
SSL D NVY---LEYN T RDQL
SSL D NVY---LEYN A RDQL
7,2 × ratiometric changes
SSL D NVY---LEYN S RDQL
8,0 × ratiometric changes
Part C: Sensors developed after cpS2 random mutagenesis
SSL E NVY---LEYN T RDQL
12.0 × increase fast maturation
SSL E NVY---LEYN S RDQL
16.5 × increase
2. Adapting the chromophore environment within a Ca2+sensor
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 , combined with cpFP described in Ref.  and originating from Ratiometric Pericam . Importantly, all our sensor variants carried Phe at position 203, which was earlier shown to influence the fluorescent properties of cpFP-based indicators .
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.
3. Characterization of Case12 and Case16 in vitro
Spectral characteristics of GCaMP-like Ca2+ sensors. Excitation/emission wavelengths, fluorescence quantum yield, extinction coefficient and pH stability in the presence of Ca2+are shown
Excitation/emission wavelengths, nm
Extinction coefficient, M-1cm-1
Relative brightness a
pH stability (pKa)
Kd for Ca2+, μM
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 . 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 Kd values for Ca2+ binding at pH 7.4, using Molecular Probes assay (Calcium Calibration Buffer Kit #1). The apparent Kd 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 [16–19].
4. Characterization of Case12 and Case16 in living cells
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)  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.
Deeper understanding of cpFP-based sensors mechanics would contribute significantly to the development of "ideal" cpFP-based sensors, characterized with high brightness, dynamic range, pH- and photo-stability. We believe that proposed spatial arrangement of cpFP-based sensors (Figure 1B) is close to the reality and that this model will be helpful for the further development of cpFP-based sensors for various analytes and cellular signaling events. However, solution of the crystal structure for one of the cpFP-based sensors remains an actual task in this respect.
Developed high dynamic range sensors Case12 and Case16 set a new record among cpFP-based and, in general, among any GFP-based sensors reported up to date . The most contrasting of the GFP-based FRET-indicators, Ca2+ sensor YC3.60 , reaches a 6.6-fold ratiometric fluorescence changes. The most contrasting of the reported cpFP-based sensors, Flash-pericam, reaches a 8-fold increase of fluorescence , whilst yielding to Case12 and Case16 in respect of pH-stability and signal brightness (Table 2). The combined advantages should provide reliable monitoring of Ca2+ signaling and suggest that Case12 and Case16 will become very popular research tools both in scientific studies and HTS assays and might be able to replace some of the currently used chemical Ca2+ probes.
Cloning and gene construction
Sensor constructs generation and site directed mutagenesis were performed by overlap-extension PCR , with primers containing the appropriate target substitutions. The cDNA coding the circularly permuted green fluorescent protein was amplified from the HyPer sensor . 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).
Protein expression and in vitrospectroscopy
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 ). 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).
Primary cultures of cortical neurons
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.
Imaging of fura-2FF and Case12 signals
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.
Experimental procedures with primary cultures of cortical neurons
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.
green fluorescent protein
Aequorea victoria green fluorescent protein, EGFP, enhanced green fluorescent protein
circularly permuted fluorescent protein
fluorescence resonance energy transfer, NGF, nerve growth factor
artificial cerebro-spinal fluid.
We gratefully acknowledge careful reading of the manuscript and continuous input and support to the project by Lukas Leder and Felix Freuler. We thank Prof. Michael I. Kotlikoff (Cornell University, Ithaca, NY) and Dr. Junichi Nakai (RIKEN Brain Science Institute, Saitama, Japan) for the kind gift of GCaMP2. This work was supported by a research grant from Novartis Pharma AG, Basel Switzerland as well as by grants from Molecular and Cell Biology Program RAS, EC FP-6 Integrated Project LSHG-CT-2003-503259, RFBR grant 07-04-12189-obr and the National Institutes of Health (GM070358). D.M.C. is supported by Grants of the President of Russian Federation MK-8236.2006.4 and Russian Science Support Foundation. Supported by a grant to SS from the Swedish Institute for exchange between Sweden and Russia. SS holds a senior scientist position from the Swedish research council and was also supported by project grants from the Swedish research council and the Swedish cancer society. JGL was supported by a postdoctoral fellowship from the Wenner-Gren foundations.
- Griesbeck O: Fluorescent proteins as sensors for cellular functions. Curr Opin Neurobiol. 2004, 14: 636-641. 10.1016/j.conb.2004.08.002.View ArticleGoogle Scholar
- Miyawaki A: Visualization of the spatial and temporal dynamics of intracellular signaling. Dev Cell. 2003, 4: 295-305. 10.1016/S1534-5807(03)00060-1.View ArticleGoogle Scholar
- Chudakov DM, Lukyanov S, Lukyanov KA: Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 2005, 23: 605-613. 10.1016/j.tibtech.2005.10.005.View ArticleGoogle Scholar
- Pologruto TA, Yasuda R, Svoboda K: Monitoring neural activity and [Ca2+] with genetically encoded Ca2+ indicators. J Neurosci. 2004, 24: 9572-9579. 10.1523/JNEUROSCI.2854-04.2004.View ArticleGoogle Scholar
- Palmer AE, Tsien RY: Measuring calcium signaling using genetically targetable fluorescent indicators. Nat Protoc. 2006, 1: 1057-1065. 10.1038/nprot.2006.172.View ArticleGoogle Scholar
- Baird GS, Zacharias DA, Tsien RY: Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci U S A. 1999, 96: 11241-11246. 10.1073/pnas.96.20.11241.View ArticleGoogle Scholar
- Nagai T, Sawano A, Park ES, Miyawaki A: Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci U S A. 2001, 98: 3197-3202. 10.1073/pnas.051636098.View ArticleGoogle Scholar
- Nakai J, Ohkura M, Imoto K: A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol. 2001, 19: 137-141. 10.1038/84397.View ArticleGoogle Scholar
- Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY: Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001, 276: 29188-29194. 10.1074/jbc.M102815200.View ArticleGoogle Scholar
- Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S: Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods. 2006, 3: 281-286. 10.1038/nmeth866.View ArticleGoogle Scholar
- Kawai Y, Sato M, Umezawa Y: Single color fluorescent indicators of protein phosphorylation for multicolor imaging of intracellular signal flow dynamics. Anal Chem. 2004, 76: 6144-6149. 10.1021/ac040037s.View ArticleGoogle Scholar
- Tallini YN, Ohkura M, Choi BR, Ji G, Imoto K, Doran R, Lee J, Plan P, Wilson J, Xin HB, Sanbe A, Gulick J, Mathai J, Robbins J, Salama G, Nakai J, Kotlikoff MI: Imaging cellular signals in the heart in vivo: Cardiac expression of the high-signal Ca2+ indicator GCaMP2. Proc Natl Acad Sci U S A. 2006, 103: 4753-4758. 10.1073/pnas.0509378103.View ArticleGoogle Scholar
- Patterson GH, Lippincott-Schwartz J: A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 2002, 297: 1873-1877. 10.1126/science.1074952.View ArticleGoogle Scholar
- Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA: Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol. 2004, 22: 1435-1439. 10.1038/nbt1025.View ArticleGoogle Scholar
- Yang F, Moss LG, Phillips GN: The molecular structure of green fluorescent protein. Nat Biotechnol. 1996, 14: 1246-1251. 10.1038/nbt1096-1246.View ArticleGoogle Scholar
- Pozzan T, Rizzuto R, Volpe P, Meldolesi J: Molecular and cellular physiology of intracellular calcium stores. Physiol Rev. 1994, 74: 595-636.Google Scholar
- Clapham DE: Calcium signaling. Cell. 1995, 80: 259-268. 10.1016/0092-8674(95)90408-5.View ArticleGoogle Scholar
- Augustine GJ, Charlton MP, Smith SJ: Calcium action in synaptic transmitter release. Annu Rev Neurosci. 1987, 10: 633-693. 10.1146/annurev.ne.10.030187.003221.View ArticleGoogle Scholar
- Llinas R, Sugimori M, Silver RB: The concept of calcium concentration microdomains in synaptic transmission. Neuropharmacology. 1995, 34: 1443-1451. 10.1016/0028-3908(95)00150-5.View ArticleGoogle Scholar
- Turner RW, Maler L, Deerinck T, Levinson SR, Ellisman MH: TTX-sensitive dendritic sodium channels underlie oscillatory discharge in a vertebrate sensory neuron. J Neurosci. 1994, 14: 6453-6471.Google Scholar
- Khodorov B: Glutamate-induced deregulation of calcium homeostasis and mitochondrial dysfunction in mammalian central neurones. Prog Biophys Mol Biol. 2004, 86: 279-351. 10.1016/j.pbiomolbio.2003.10.002.View ArticleGoogle Scholar
- Nicholls DG, Budd SL: Mitochondria and neuronal survival. Physiol Rev. 2000, 80: 315-360.Google Scholar
- Wu ML, Chen JH, Chen WH, Chen YJ, Chu KC: Novel role of the Ca(2+)-ATPase in NMDA-induced intracellular acidification. Am J Physiol. 1999, 277: C717-27.Google Scholar
- Pinelis VG, Khodorov BI, Fajuk D, Zagulova D, Andreeva NA, Uvarova T, Khaspekov LG, Golovina VA, Viktorov IV: A persistent calcium-dependent decrease of cytoplasmic pH in cultured nerve cells induced by toxic glutamate treatment. Biologicheskie Membrany. 1992, 9: 1049-1051.Google Scholar
- Brocard JB, Tassetto M, Reynolds IJ: Quantitative evaluation of mitochondrial calcium content in rat cortical neurones following a glutamate stimulus. J Physiol. 2001, 531: 793-805. 10.1111/j.1469-7793.2001.0793h.x.View ArticleGoogle Scholar
- Kiedrowski L, Costa E: Glutamate-induced destabilization of intracellular calcium concentration homeostasis in cultured cerebellar granule cells: role of mitochondria in calcium buffering. Mol Pharmacol. 1995, 47: 140-147.Google Scholar
- Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A: Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins. Proc Natl Acad Sci U S A. 2004, 101: 10554-10559. 10.1073/pnas.0400417101.View ArticleGoogle Scholar
- Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR: Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989, 77: 51-59. 10.1016/0378-1119(89)90358-2.View ArticleGoogle Scholar
- Patterson G, Day RN, Piston D: Fluorescent protein spectra. J Cell Sci. 2001, 114: 837-838.Google Scholar
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