Fluorescent Biosensor for Quantitative Real-time Measurements of Inositol 1,4,5-Trisphosphate in Single Living Cells

Abstract

The second messenger inositol 1,4,5-trisphosphate (IP3) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca2+ signals involved in the regulation of many essential physiological processes. Here we describe the development of “LIBRA”, a novel ratiometric fluorescent IP3 biosensor that allows for the quantitative monitoring of intracellular IP3 concentrations in single living cells in real time. LIBRA consists of the IP3-binding domain of the rat type 3 IP3 receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP3 and unaffected by concentrations of Ca2+ and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP3 and Ca2+ concentrations in SH-SY5Y cells following acetylcholine stimulation. The second messenger inositol 1,4,5-trisphosphate (IP3) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca2+ signals involved in the regulation of many essential physiological processes. Here we describe the development of “LIBRA”, a novel ratiometric fluorescent IP3 biosensor that allows for the quantitative monitoring of intracellular IP3 concentrations in single living cells in real time. LIBRA consists of the IP3-binding domain of the rat type 3 IP3 receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP3 and unaffected by concentrations of Ca2+ and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP3 and Ca2+ concentrations in SH-SY5Y cells following acetylcholine stimulation. Many plasma membrane receptors act by stimulating the phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; Ach, acetylcholine; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; EGFP, enhanced GFP; HBSS, Hanks' balanced salt solution; ICM, intracellular-like medium; IP2, inositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; 1,3,4-IP3, inositol 1,3,4-trisphosphate; PHD, pleckstrin homology domain; [Ca2+]i, cytosolic Ca2+ concentration; [IP3]i, cytosolic IP3 concentration; CFP, cyan fluorescent protein; ECFP, enhanced CFP; YFP, yellow fluorescent protein; EYFP, enhanced YFP; LIBRA luminous inositol trisphosphate-binding domain for ratiometric analysis.1The abbreviations used are: PLC, phospholipase C; Ach, acetylcholine; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; EGFP, enhanced GFP; HBSS, Hanks' balanced salt solution; ICM, intracellular-like medium; IP2, inositol 4,5-bisphosphate; IP3, inositol 1,4,5-trisphosphate; IP4, inositol 1,3,4,5-tetrakisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; 1,3,4-IP3, inositol 1,3,4-trisphosphate; PHD, pleckstrin homology domain; [Ca2+]i, cytosolic Ca2+ concentration; [IP3]i, cytosolic IP3 concentration; CFP, cyan fluorescent protein; ECFP, enhanced CFP; YFP, yellow fluorescent protein; EYFP, enhanced YFP; LIBRA luminous inositol trisphosphate-binding domain for ratiometric analysis.-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the intracellular messenger inositol 1,4,5-trisphosphate (IP3) (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6134) Google Scholar). IP3 in turn releases Ca2+ from intracellular stores via IP3 receptors (IP3Rs) resulting in the activation of a variety of Ca2+-dependent processes. IP3-dependent Ca2+ responses can exhibit complex spatial and temporal patterns, and a number of competing models have been proposed to account for them (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6134) Google Scholar, 2Nash M.S. Young K.W. Challiss R.A.J. Nahorski S.R. Nature. 2001; 413: 381-382Crossref PubMed Scopus (117) Google Scholar, 3Taylor C.W. Thorn P. Curr. Biol. 2001; 11: R352-R355Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Because of its central role in these responses, the ability to quantitatively monitor temporal changes in [IP3]i in single living cells is of vital importance to understanding these phenomena. Since IP3Rs are the natural physiological target for IP3, we reasoned that their IP3-binding domain (4Mignery G.A. Südhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar, 5Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (367) Google Scholar) might be used to construct an effective and specific intracellular IP3 detector. Here we describe such a ratiometric fluorescent biosensor that in addition allows for in situ calibration. We demonstrate the utility of this biosensor by examining the relationship between the responses of [IP3]i and [Ca2+]i in single living cells. Media—Hanks' balanced salt solution with Hepes (HBSS-H) contained 137 mm NaCl, 5.4 mm KCl, 1.3 mm CaCl2, 0.41 mm MgSO4, 0.49 mm MgCl2, 0.34 mm Na2HPO4, 0.44 mm NaH2PO4, 5.5 mm glucose, 20 mm Hepes-NaOH, pH 7.4. Intracellular-like medium (ICM) contained 125 mm KCl, 19 mm NaCl, 10 mm Hepes-KOH, pH 7.3, 1 mm EGTA, and appropriate concentrations of CaCl2 (330 μm CaCl2 for 50 nm free Ca2+) (6Tanimura A. Turner R.J. J. Biol. Chem. 1996; 271: 30904-30908Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Plasmid Construction—A CFP/YFP fusion construct was made by cutting EYFP out of pEYFP-N1 (Clontech) using BamHI and XbaI and ligating into pECFP-C1 (Clontech) cut with the same enzymes. The multiple cloning site of this vector was then removed by cutting with BspEI and BamHI and replaced with a linker generated from two synthetic oligonucleotides. The forward sequence of this linker was TCC GGA AAG CTC GAG GCA GTA AGA TCT GGC TCC GCC GAC GAT GAC GAT AAG GCC GGA TCT GTC GAC GCA GTC GGA TCC, where the reconstituted BspEI and BamHI sites have been included for clarity and the sequence has been parsed into codons. This fusion construct was referred to as pCY-N. The linker plus EYFP sequence of pCY-N was then cut out with BsrGI and ligated into pECFP-mem (Clontech) cut with the same enzyme. Finally the multiple cloning site originating from pECFP-mem was removed by cutting with Eco47III and SmaI and religating. The resultant construct, mCY, codes for ECFP preceded by the N-terminal 20 amino acids of neuromodulin (a membrane-targeting signal) and followed by the above linker and EYFP. To construct LIBRA (luminous inositol trisphosphate-binding domain for ratiometric analysis) the IP3-binding domain of the rat type 3 IP3R (amino acids 1–604) was amplified by PCR using pCB-EGFP: IP3R3 (7Morita T. Tanimura A. Nezu A. Tojyo T. Cell Calcium. 2002; 31: 59-64Crossref PubMed Scopus (10) Google Scholar) as the template and incorporating XhoI sites at either end. This sequence was then ligated into the XhoI site in the linker region of mCY using standard methods. LIBRAΔN was constructed in the same way using amino acids 227–604 of the rat type 3 IP3R (8Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar). The forward PCR primers used were ACG CAT ACT CGA GAT GAA TGA AAT GTC CAG C for LIBRA and AAG CAT ACT CGA GTT CCG GGA CCA TCT GGA G for LIBRAΔN. The same reverse primer, AGC GTA TCT CGA GCT TCC GGT TGT TGT GCA G, was used for both PCR reactions. The correctness of all constructs was confirmed by restriction digestion and sequencing. Cell Culture and Transfection—SH-SY5Y cells purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with low glucose (100 mg/ml), supplemented with 10% newborn calf serum, 584 mg/ml l-glutamine, 110 mg/ml sodium pyruvate, 100 units/ml penicillin and 100 μg/ml streptomycin. Cells were transfected with plasmids using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Measurement of Fluorescence—SH-SY5Y cells were grown on fibronectin-coated glass cover slips incorporated into an experimental chamber (6Tanimura A. Turner R.J. J. Biol. Chem. 1996; 271: 30904-30908Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cells were washed with HBSS-H and rested for at least 15 min prior to experiments. In some experiments, cells were loaded with fura-2 by incubation for 5 min at room temperature in HBSS-H containing 0.25–0.5 μm fura-2/AM (Dojin Chemicals, Kumamoto, Japan). Cells in HBSS-H or Ca2+-free HBSS-H (in which 1.3 mm CaCl2 was replaced with 0.1 mm EGTA) were stimulated by exposure to various concentration of acetylcholine (Ach) or 1 μm ionomycin as indicated. Permeabilization was performed by exposing cells to ICM containing 100 μg/ml (w/v) saponin (ICN, Cleveland, OH) for ∼1 min. Permeabilized cells were washed with ICM and then exposed to ICM containing various concentrations of IP3 and other reagents. Fluorescence images were captured using a dual-wavelength ratio imaging system (Hamamatsu Photonics, Shizuoka, Japan) consisting of a cooled CCD camera (HiSCA) and W-View optics coupled to a Nikon Diaphot 300 inverted fluorescence microscope equipped with a Nikon Fluor 40 oil immersion objective (NA 1.3). Fluorescence of LIBRA, LIBRAΔN, and mCY were monitored with excitation at 425 nm and dual-emission at 480 and 535 nm. Simultaneous monitoring of LIBRA and fura-2 was performed with sequential excitation at 380 nm (for fura-2) and 425 nm (for LIBRA) and dual-emission at 480 nm (for LIBRA) and 535 nm (for LIBRA and fura-2). Data were analyzed with AQUACOSMOS software (Hamamatsu Photonics). [Ca2+]i was calculated from the fura-2 fluorescence intensity, F, using the formula [Ca2+] = Kd(F — Fmin)/(Fmax — F), where Kd = 135 nm, and Fmin and Fmax are the values of F at zero and limitingly high [Ca2+], respectively (9Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Fmin was determined by assuming that the resting [Ca2+]i was 50 nm; Fmax was measured following the application of 1 μm ionomycin. We designed two potential IP3 biosensors (Fig. 1a and see “Experimental Procedures”) consisting of the IP3-binding domain of the rat type 3 IP3R (8Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar) fused between the well established FRET pair CFP and YFP (10Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Nature. 1997; 388: 882-887Crossref PubMed Scopus (2570) Google Scholar, 11Miyawaki A. Dev. Cell. 2003; 4: 295-305Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar) and preceded by the N-terminal 20 amino acids of neuromodulin (12Skene J.H.P. Virág I. J. Cell Biol. 1989; 108: 613-624Crossref PubMed Scopus (313) Google Scholar), a plasma membrane-targeting signal. The first of these constructs, which we refer to as LIBRA contains residues 1–604 of the rat type 3 IP3R, while LIBRAΔN contains residues 227–604. The control molecule mCY lacks the IP3-binding domain. Using confocal microscopy we found that these recombinant proteins localized to the plasma membrane and Golgi area when expressed in SH-SY5Y human neuroblastoma cells (data not shown); also ∼90% of their fluorescence was retained by the cells after permeabilization with saponin (Fig. 1b). We looked for FRET between CFP and YFP by exciting CFP at 425 nm and recording emitted fluorescence at 480 and 535 nm. When permeabilized SH-SY5Y cells transfected with LIBRA were exposed to 10 μm IP3 an increase in the 480 nm signal (∼4.5%) and a parallel decrease in the 535 nm signal (∼3.6%) were observed within ∼1 s (Fig. 1c). By contrast neither LIBRAΔN nor mCY showed any detectable fluorescence changes in response to 10 μm IP3 (Fig. 1d). The CFP donor quenching of LIBRA that was relieved by YFP acceptor photobleaching (13Miyawaki A. Tsien R.Y. Methods Enzymol. 2000; 327: 472-500Crossref PubMed Scopus (374) Google Scholar) was ∼3.9% in the presence of 10 μm IP3 and ∼8.6% in its absence, confirming that the FRET efficiency of LIBRA is decreased in its IP3-bound form. It has been demonstrated that residues 226–576 of the mouse type 1 IP3R constitute the essential “core” region required for high affinity IP3 binding and that the N-terminal 225 residues act to suppress the IP3 binding affinity of the core (14Yoshikawa F. Uchiyama T. Iwasaki H. Tomomori S.C. Tanaka T. Furuichi T. Mikoshiba K. Biochem. Biophys. Res. Commun. 1999; 257: 792-797Crossref PubMed Scopus (42) Google Scholar). Interestingly, LIBRAΔN, which lacks this N-terminal suppressor region, shows no IP3-dependent changes in fluorescence. Accordingly we speculate that the conformational change underlying the IP3-dependent LIBRA FRET signal could be related to an effect of IP3 on the interaction between the core and suppressor regions. Changes in the LIBRA 480/535 nm emission ratio (ΔRatio) were highly sensitive to [IP3] and selective for 1,4,5-IP3versus other inositol phosphates (Fig. 2). ΔRatio had a monophasic dependence on [IP3] with apparent dissociation constant (Kd) of 404 nm and Hill coefficient (n) of 1.118 (Fig. 2b). The Kd for adenophostin A, a more potent agonist for the IP3R than IP3 (15Takahashi M. Tanzawa K. Takahashi S. J. Biol. Chem. 1994; 269: 369-372Abstract Full Text PDF PubMed Google Scholar), was 65 nm and we estimate that inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 4,5-bisphosphate (IP2), and inositol 1,3,4-trisphosphate (1,3,4-IP3) bind to LIBRA with Kd values of ∼15, ∼40, and »10 μm, respectively (Fig. 2b). It is known that the activity of the IP3R is modulated by Ca2+ (16Bezprozvanny I. Watras J. Ehrlich B.E. Nature. 1991; 351: 751-754Crossref PubMed Scopus (1418) Google Scholar, 17Iino M. Endo M. Nature. 1992; 360: 76-78Crossref PubMed Scopus (242) Google Scholar) and ATP (18Ferris C.D. Huganir R.L. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2147-2151Crossref PubMed Scopus (149) Google Scholar), although their effects are not mediated by the IP3-binding domain (5Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (367) Google Scholar, 19Miyakawa T. Mizushima A. Hirose K. Yamazawa A. Bezprozvanny I. Kurosaki T. Iino M. EMBO J. 2001; 20: 1674-1680Crossref PubMed Scopus (114) Google Scholar). In control experiments we have confirmed that both Ca2+ (0–1 μm) and ATP (0–3 mm) have no effect on LIBRA fluorescence or on the ΔRatio seen in response to IP3 (Fig. 2c). We also found that, while changes in pH affected the basal emission ratio as a result of the well known effects of pH on YFP fluorescence (13Miyawaki A. Tsien R.Y. Methods Enzymol. 2000; 327: 472-500Crossref PubMed Scopus (374) Google Scholar), the ΔRatio elicited by IP3 was unaffected over the pH range 7.0–7.6 (Fig. 2d). Taken together the above results show that ΔRatio (at constant pH) is a sensitive and specific measure of [IP3]. We next used the ΔRatio of LIBRA to follow the dynamics of [IP3]i after muscarinic stimulation of intact SH-SY5Y cells (Fig. 3a). In the presence of extracellular Ca2+, application of 10 or 100 μm Ach increased ΔRatio in >80% of LIBRA-expressing cells, and 1 μm Ach elicited a response in ∼60% of the cells tested. Increasing [Ca2+]i with ionomycin had no effect on ΔRatio (Fig. 3, a and e) and the application of 100 μm Ach caused no changes in fluorescence in either mCY-expressing cells or in cells loaded with the intracellular pH indicator BCECF (data not shown), confirming the specificity of the LIBRA response. The PLC inhibitor U73122 (20Smith R.J. Sam L.M. Justen J.M. Bundy G.L. Bala G.A. Bleasdale J.E. J. Pharmacol. Exp. Ther. 1990; 253: 688-697PubMed Google Scholar) completely blocked the Ach-induced increase in ΔRatio but had no effect on the resting emission ratio (Fig. 3b) suggesting that the resting [IP3]i in these cells is below the detectable range of LIBRA. The magnitude of the LIBRA ΔRatio response increased with [Ach] reaching a maximum 2–5 min after stimulation (Fig. 3, a and c). As discussed in more detail below the time scale of these responses is much longer than the onset of Ach-induced Ca2+ spikes in this cell type (5–20 s). Peak ΔRatio values were similar in the presence and absence of extracellular Ca2+ (Fig. 3c); however, significant differences were seen in the time courses of these responses (Fig. 3a). In the presence of Ca2+,in 20 out of 32 responding cells, ΔRatio rose to a maximum while in the cells ΔRatio rose to a then to a that was of the response. In in the absence of Ca2+, out of responding cells showed a in ΔRatio in response to in the cells ΔRatio rose to a then to a to resting In both experimental the application of Ach elicited a in [Ca2+]i using which then to a above in the presence of extracellular Ca2+ and to in its absence (data not The in ΔRatio in the presence of extracellular Ca2+ and the in its absence (Fig. a role for a in [Ca2+]i in increased [IP3] To for effects of LIBRA on Ach-induced Ca2+ we the for the onset of Ca2+ spikes after Ach stimulation. the application of 1 μm Ach in the absence of extracellular Ca2+ this was s = in LIBRA-expressing cells and s = in cells. Since any of intracellular LIBRA have been to increase this to the Ca2+ these results are with the that LIBRA has any effect on intracellular IP3 experimental To the relationship between the Ach-induced [IP3]i response and Ca2+ from intracellular we monitored the fluorescence of both LIBRA and the [Ca2+]i indicator fura-2 in the same cells in Ca2+-free In experiments where cells were exposed to two of a low the second application of Ach elicited a significant but increase in [IP3]i than the first and was by or no increase in [Ca2+]i (Fig. with that [IP3] to a of Ca2+ S. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). In in similar experiments in Ca2+ containing medium both of Ach in in both [IP3]i and [Ca2+]i (data not Interestingly, at Ach concentrations μm in some at 1 μm) the of the [IP3]i response after the in [Ca2+]i that [IP3]i was as [Ca2+]i (Fig. 3, and results were observed in the presence of extracellular Ca2+ (data not low [Ach] a application of a [Ach] in Ca2+ (Fig. that this temporal dissociation of the [IP3]i and [Ca2+]i responses is not to the of intracellular Ca2+ be required to this relationship between [IP3]i and By assuming that the response of the LIBRA ΔRatio to IP3 is the same in intact and permeabilized cells, can the above LIBRA signals to [IP3]i using the in with this we have confirmed that the increase in ΔRatio to of a concentration of IP3 into intact LIBRA-expressing cells is not from the increase in ΔRatio to IP3 in permeabilized cells (data not we estimate that the [IP3]i required to a Ca2+ in SH-SY5Y cells is nm. This is to the concentration of IP3 found to be required for [Ca2+]i spikes in I. I. J. 1992; PubMed Google Scholar). We also estimate that the [IP3]i by and 100 μm Ach in SH-SY5Y cells are and nm, results demonstrate the utility of LIBRA to follow changes in [IP3]i in intact cells in real time. LIBRA has over a for monitoring [IP3]i that the intracellular of a protein consisting of the pleckstrin homology domain of fused to K. S. M. H. Iino M. 1999; PubMed Scopus Google Scholar). This was designed to changes in membrane S. T. Curr. Biol. Full Text Full Text PDF PubMed Google Scholar, P. T. J. Cell Biol. PubMed Scopus Google Scholar) but was also to be sensitive to [IP3]i M.S. Young K.W. Challiss R.A.J. Nahorski S.R. Nature. 2001; 413: 381-382Crossref PubMed Scopus (117) Google Scholar, K. S. M. H. Iino M. 1999; PubMed Scopus Google Scholar). experiments have confirmed that can changes in both IP3 and and that its with J. R. P. T. K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Watras J. L.M. J. Cell Biol. 2003; PubMed Scopus Google Scholar). In addition to these for J. R. P. T. K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Watras J. L.M. J. Cell Biol. 2003; PubMed Scopus Google Scholar) has not been to the signal. The temporal dynamics of G protein coupled Ca2+ responses have been to on the effects of cytosolic and (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6134) Google Scholar, A. Turner R.J. J. Biol. Chem. 1996; 271: 30904-30908Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 17Iino M. Endo M. Nature. 1992; 360: 76-78Crossref PubMed Scopus (242) Google Scholar, A. Turner R.J. J. Cell Biol. 1996; PubMed Scopus Google Scholar) as well as on to IP3 Young K.W. Young J.M. M.J. Biochem. J. 1996; PubMed Scopus Google Scholar). In addition, the of and proteins on IP3 M. S.H. J. S.K. S. J. Cell Biol. 2003; PubMed Scopus Google Scholar). We that the ability to quantitatively monitor [IP3]i with LIBRA and related ratiometric biosensors in the of the of these and other effects on the generation of spatiotemporally complex Ca2+