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¹ Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
² Division of Molecular Neurobiology, Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
³ Calcium Oscillation Project, International Cooperative Research Project—Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan
⁴ Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, Saitama 351-0198, Japan
⁵ Misaki Marine Biological Station, Graduate School of Science, University of Tokyo, Kanagawa, 238-0225, Japan

Correspondence to Takayuki Michikawa: takamich{at}ims.u-tokyo.ac.jp; or Katsuhiko Mikoshiba: mikosiba{at}ims.u-tokyo.ac.jp

Top Abstract Introduction Results Discussion Materials and methods References

 
We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP₃) sensors that do not severely interfere with intracellular Ca²⁺ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP₃ and Ca²⁺ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP₃ started to increase at a relatively constant rate before the pacemaker Ca²⁺ rise, and the subsequent abrupt Ca²⁺ rise was not accompanied by any acceleration in the rate of increase in IP₃. Cytosolic [IP₃] did not return to its basal level during the intervals between Ca²⁺ spikes, and IP₃ gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca²⁺ oscillations. These results indicate that the Ca²⁺-induced regenerative IP₃ production is not a driving force of the upstroke of Ca²⁺ spikes and that the apparent IP₃ sensitivity for Ca²⁺ spike generation progressively decreases during Ca²⁺ oscillations.

	Introduction

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An increase in cytosolic [Ca²⁺] ([Ca²⁺]_c) is a ubiquitous intracellular signal that controls various cellular processes, including fertilization, proliferation, development, learning and memory, contraction, and secretion (Berridge et al., 2000). [Ca²⁺]_c rises evoked by extracellular stimuli are often observed in the form of pulsatile Ca²⁺ spikes that result from the transient opening of Ca²⁺ channels located either in the plasma membrane or on the cytosolic Ca²⁺ stores (Berridge and Dupont, 1994). The spatial counterpart of Ca²⁺ spikes are Ca²⁺ waves, which are produced when an initial localized [Ca²⁺]_c elevation leads to the propagation of the rise in [Ca²⁺]_c throughout the cytoplasm (Lechleiter et al., 1991; Thomas et al., 1991). The frequency of the occurrence of Ca²⁺ spikes is correlated with the stimulus intensity (Woods et al., 1986; Berridge, 1988; Jacob et al., 1988; Prentki et al., 1988), and the time course of an individual Ca²⁺ spike depends on the type of receptor stimulated but not on the stimulus intensity (Cobbold et al., 1991; Thomas et al., 1991), indicating that the information of both the stimulus species and its intensity is encoded within the temporal pattern of cytosolic Ca²⁺.

Stimulus-induced cytosolic Ca²⁺ spikes usually form as a result of an initial slow pacemaker rise in [Ca²⁺]_c followed by a rapid rise in [Ca²⁺]_c (Jacob et al., 1988; Thomas et al., 1991; Bootman and Berridge, 1996). The rate of the rapid [Ca²⁺]_c rise remains relatively constant regardless of the stimulus intensity (Cobbold et al., 1991; Thomas et al., 1991), suggesting that a regenerative process is involved in the generation of the abrupt upstroke (Thomas et al., 1991). Such regenerative processes require a positive-feedback element (Meyer and Stryer, 1991), and some molecules that are critical to the regenerative process have been proposed. PLC catalyzes the formation of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol from phosphatidylinositol 4,5-bisphosphate and has been hypothesized to act as a positive-feedback element (Meyer and Stryer, 1988; Harootunian et al., 1991) because its activity is stimulated by cytosolic Ca²⁺ (Rebecchi and Pentyala, 2000). According to their hypothesis, the positive-feedback regulation of PLC by cytosolic Ca²⁺ allows generation of IP₃ spikes that result in Ca²⁺ spikes through activation of the IP₃ receptor (IP₃R)/IP₃-gated Ca²⁺ release channel (Meyer and Stryer, 1988). Another hypothesis is that the positive-feedback regulation is attributable to the intrinsic property of the IP₃R. The biphasic dependency of channel activity on Ca²⁺ (Iino, 1990) may create the rapid upstroke of Ca²⁺ spikes even at constant levels of IP₃ (Wakui et al., 1989; Osipchuk et al., 1990; Wakui et al., 1990; Dupont et al., 1991; De Young and Keizer, 1992; Hajnoczky and Thomas, 1997). These mechanisms for the generation of Ca²⁺ spikes, however, are not necessarily exclusive, and combined models are also plausible.

Observation of cytosolic IP₃ dynamics during Ca²⁺ spikes should help us better understand the mechanism responsible for the Ca²⁺ spike generation (Meyer and Stryer, 1991). The GFP-tagged pleckstrin homology domain (PHD) of PLC-1, which interacts with phosphatidylinositol 4,5-bisphosphate and/or IP₃, has been used to monitor IP₃ production and its oscillatory translocation from plasma membrane to the cytosol synchronously with Ca²⁺ oscillation was observed in MDCK cells (Hirose et al., 1999), suggesting that each Ca²⁺ spike accompanies IP₃ production. However, there is no simple correlation between its translocation from the plasma membrane to the cytosol and actual cytosolic [IP₃] ([IP₃]_c van der Wal et al., 2001; Xu et al., 2003; Irvine, 2004). Fluorescent IP₃ sensors based on the IP₃ binding domain of IP₃R have recently been developed (Tanimura et al., 2004; Sato et al., 2005; Remus et al., 2006), but no one has ever used them to investigate the mechanism underlying the Ca²⁺ spike generation in living cells.

In the present study, we developed low IP₃ binding affinity, high signal-to-noise ratio cytosolic IP₃ sensors based on the IP₃ binding domain of mouse type 1 IP₃R (IP₃R1) and used them to analyze the mechanism responsible for the generation of Ca²⁺ oscillations. Simultaneous imaging of [Ca²⁺]_c and [IP₃]_c in living cells exposed to extracellular stimuli provided us with a novel insight into the mechanism of generation of intracellular Ca²⁺ spikes.

	Results

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Development of cytosolic IP₃ sensors based on the IP₃ binding domain of IP₃R1
To develop cytosolic IP₃ sensors, we constructed tandem fusion proteins of a variant of yellow fluorescent protein, Venus (Nagai et al., 2002); an IP₃ binding domain of IP₃R1 (Miyawaki et al., 1991; Yoshikawa et al., 1996; Bosanac et al., 2002); and an enhanced cyan fluorescent protein (ECFP; Fig. 1 A ). We used amino acid residues 224–569, 224–575, 224–579, 224–584, and 224–604 of mouse IP₃R1 as an IP₃ binding motif and found that all fusion proteins except the protein composed of residues 224–569 showed IP₃-dependent decrease of fluorescence resonance energy transfer (FRET) between Venus and ECFP (Fig. 1 B). The largest FRET change was obtained in the fusion protein composed of residues 224–575 of mouse IP₃R1, and the 475-nm (ECFP) to 525-nm (Venus) emission ratio of the fusion protein increased by 25.1 ± 8.0% (n = 3) after the addition of 60 µM IP₃ into cell lysates. We designated this fusion protein as IP₃R-based IP₃ sensor 1 (IRIS-1). The emission ratio of IRIS-1 changed depending on the concentration of IP₃ applied, but its apparent IP₃ sensitivity (K_d = 549 ± 62 nM; n = 3) was significantly lower than those of fusion proteins composed of residues 224–604 (K_d = 107 ± 41 nM; n = 3), 224–584 (K_d = 105 ± 0 nM; n = 3), and 224–579 (K_d = 95 ± 38 nM; n = 3; Fig. 1 C).

View larger version (18K): [in this window] [in a new window]   Figure 1. IP3 sensor proteins based on the IP3 binding domain of mouse IP3R1. (A) A basic design for IP3 sensors. (B) ECFP/Venus emission ratio changes of IP3 sensors in COS7 lysates after the addition of 60 µM of IP3. (C) Apparent IP3 affinity of IP3 sensors composed with amino acid residues 224–575 (circle), 224–579 (triangle), 224–584 (square), and 224–604 (inverse triangle) in COS7 lysates. Data were obtained from three independent measurements. Emission spectra of purified IRIS-1 (D) and IRIS-1–Dmut (E) excited at 420 nm with 0 µM (broken line) and 100 µM (solid line) of IP3. Measurements were performed at 20°C in buffer A (10 mM Hepes, pH 7.2, 100 mM NaCl, 1 mM 2-mercaptoethanol, and 0.5% NP-40) containing 1 mM EDTA. (F) Specificity of IRIS-1 against IP3 (circle) and its natural metabolites, 1,3,4,5 IP4 (triangle) and 1,4 IP2 (square). Measurements were performed at 20°C in buffer A containing 1 mM EDTA. (G) Ca2+ sensitivity of IRIS-1. Emission of IRIS-1 was measured in buffer A containing 1 mM HEDTA (circle) or 1 µM free Ca2+ (square). The free Ca2+ concentration was adjusted as described elsewhere (Michikawa et al., 1999). (H) pH sensitivity of IRIS-1. Three buffers with different pH (circle, pH 7.0; square, pH 7.4; triangle, pH 7.8) were prepared based on buffer A containing 1 mM EDTA. Error bars correspond to the SD (n = 3).

IRIS-1 was uniformly distributed within the cytosol of intact HeLa cells (Fig. 2 A), and 80% of IRIS-1 was released from the cells after 5 min of treatment with 0.1% saponin (Fig. 2, A and B ). ECFP-tagged IP₃R1, which is an ER resident protein, was hardly released from the cells with the same treatment (Fig. 2, A and B). These results indicate that IRIS-1 is mainly localized in the cytosol of HeLa cells. To monitor cytosolic IP₃ and Ca²⁺ dynamics simultaneously, Indo-1 was loaded into IRIS-1–expressing HeLa cells. Fig. 2 D shows FRET changes of IRIS-1 accompanied by Ca²⁺ transients (Fig. 2 C) elicited by sequential stimulations with 1, 5, and 10 µM of histamine. We did not detect FRET changes in IRIS-1–Dmut–expressing cells even when Ca²⁺ transients were observed (Fig. 2, E and F), and the expression level of IRIS-1–Dmut was indistinguishable from that of IRIS-1 (Fig. 2 G). FRET changes of IRIS-1 were completely blocked by the addition of 10 µM of PLC inhibitor U73122 but not by its inactive analogue U73343 in (Fig. S1, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200512141/DC1). These results indicate that IRIS-1 can monitor [IP₃]_c changes in living cells.

View larger version (25K): [in this window] [in a new window]   Figure 2. Characterization of IRIS-1 expressed in HeLa cells. (A) Intracellular localization of IRIS-1 and ECFP-tagged mouse IP3R1 shown by the ECFP fluorescence excited at 425–445 nm. Cells were treated with a solution containing 0.1% saponin, 80 mM Pipes, pH 7.2, 1 mM MgCl2, 1 mM EGTA, and 4% polyethylen glycol for 5 min at room temperature and were washed with balanced salt solution for 10 min. Bars, 10 µm. (B) Relative fluorescence intensity of IRIS-1 (n = 20) and ECFP-tagged IP3R1 (n = 6) after the saponin treatment. Error bars indicate SD. (C–F) Time courses of emission changes of Indo-1 (C) and IRIS-1 (D) in a single HeLa cell sequentially stimulated with 1, 5, and 10 µM of histamine (horizontal bars). The same experiments were performed in IRIS-1–Dmut–expressing HeLa cells (E and F). Three different color plots represent data from three cells in the same viewing field (C–F). ECFP/Venus emission ratio (IRISs and C/V-PHD) and 420–440/460–510-nm emission ratio (Indo-1) were defined as R, and R was defined as R – Rbase, where Rbase is the basal level of R, in this and the following figures. (G) Expression levels of IRIS-1 (lane 1) and IRIS-1–Dmut (lane 2) in HeLa cells assessed by Western blot analysis using an anti-GFP antibody. Similar results were observed in three independent experiments. Molecular mass markers are shown on the left (x10–3).

View larger version (24K): [in this window] [in a new window]   Figure 3. Effects of exogenously expressed proteins on Ca2+ oscillation frequency in mGluR5a-expressing HeLa cells. (A) Emission ratio change of Indo-1 signals in cells stimulated with 100 µM of glutamate (horizontal bars). (B) Histograms of Ca2+ oscillation frequency in mGluR5a-expressing cells stimulated with 100 µM of glutamate. (C) Western blot analysis of cell lysates prepared from HeLa cells transfected with mGluR5a alone (lane 2), mGluR5a plus C/V-PHD (lane 3), mGluR5a plus IRIS-1 (lane 4), or mGluR5a plus IRIS-1–Dmut (lane 5). Nontransfected cells were used as a control (lane 1). An anti-mGluR5 antibody was used. Molecular mass markers are shown on the left (x10–3).

View larger version (22K): [in this window] [in a new window]   Figure 4. IP3 dynamics during Ca2+ oscillations. (A) HeLa cells expressing mGluR5a were stimulated with 100 µM of glutamate (horizontal bars). Similar results were observed in 28 out of 29 cells (IRIS-1), 15 out of 17 cells (IRIS-1–Dmut), and 3 out of 12 cells (C/V-PHD). (B) IRIS-1–expressing HeLa cells were stimulated with 3 µM of histamine (horizontal bar). Similar results were observed in 15 cells. (C) The IP3 concentrations at the time of the onset of each Ca2+ spike are shown over the Ca2+ spike number. Results from mGluR5a-expressing HeLa cells stimulated with 100 µM of glutamate (closed circle) and HeLa cells stimulated with 3 µM of histamine (open circle) are shown. Values are averaged for 14–24 measurements (closed circle) and 6–15 measurements (open circle). Error bars correspond to the SD.

We did not detect IP₃ spikes, i.e., rapid upstrokes of [IP₃]_c with a constant amplitude followed by a gradual decrease of [IP₃]_c to its basal level, even when Ca²⁺ spikes were triggered by glutamate (Fig. 4 A) or histamine application (Fig. 4 B). However, if the IP₃ sensor was saturated with IP₃ in the cells and/or the FRET change of IRIS-1 upon IP₃ binding was too slow to detect [IP₃]_c changes, we would have missed the IP₃ spikes, so we conducted the following experiments to investigate these possibilities. IRIS-1–expressing HeLa cells were permeabilized with 60 µM ß-escin for 3 min and then exposed to solutions containing various concentrations of IP₃. IRIS-1 exhibited a maximal response of 28.0% after addition of an excess amount of IP₃ under the conditions used (Fig. S1 C). The maximal response observed was greater than that observed in intact cells stimulated with 100 µM glutamate (20%; Fig. 4 A) and 3 µM histamine (7%; Fig. 4 B), indicating that the dynamic range of changes in the IRIS-1 signals is greater than the changes in [IP₃]_c evoked by the stimuli when the resting level of [IP₃]_c is sufficiently low (<100 nM). The possibility of saturation of the IRIS-1 signals was further investigated with IRIS-1.2, a low-affinity mutant of IRIS-1 in which lysine 249 is replaced by glutamine (K249Q). IRIS-1.2 exhibited approximately sevenfold lower affinity for IP₃ than IRIS-1 (Fig. S1 D). FRET signals observed with IRIS-1.2 revealed temporal patterns similar to those observed with IRIS-1 during Ca²⁺ oscillations evoked by mGluR5a stimulation (Fig. S1, E and F). The IRIS-1.2 signals rapidly rose after the addition of glutamate, and they remained at the elevated level and underwent fluctuations (but not baseline spikes) during Ca²⁺ oscillations. These results indicate that the IRIS-1 signals in intact cells were not saturated when the mGluR5a-expressing HeLa cells were stimulated with 100 µM glutamate. The rate of changes in IRIS-1 signals was evaluated directly by measurements of the fluorescent changes of IRIS-1 after rapid mixing with IP₃ using stopped-flow fluorescence spectrometry (see the supplemental text and Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200512141/DC1). The analysis of the kinetics of the fluorescence changes indicates that there are two conformations of IRIS-1 with a different FRET efficiency and only IRIS-1 with a low FRET efficiency is able to bind to IP₃ (see the supplemental text). The kinetic parameters were estimated from both the kinetics data (Fig. S2 B) and the equilibrium data (Fig. S2 C) and were used to calculate the FRET changes during and after the addition of a brief pulse of IP₃. The resting level of [IP₃]_c was configured to be 40 nM based on measurements in Xenopus laevis oocytes (Luzzi et al., 1998). Fig. S2 D shows the dynamics of the fraction of IRIS-1 with low FRET efficiency evoked by a 1-s pulse of various concentrations of IP₃ (from 200 nM to 12.8 µM). The fraction of IRIS-1 with low FRET efficiency increased more than twofold 1 s after the onset of the IP₃ pulses and returned to its basal level within 3 s after the IP₃ pulses with all concentrations applied (Fig. S2 D). The frequency of the Ca²⁺ oscillations in the IRIS-1–expressing cells stimulated with 100 µM glutamate was 36.9 ± 7.4 mHz (Fig. 3 B), and the mean duration of the interspike interval was 27.1 s. If [IP₃]_c actually returns to it basal level during the period of each Ca²⁺ spike, IRIS-1 would be able to monitor it. We therefore concluded that IRIS-1 possesses a sufficient temporal property to detect [IP₃]_c changes during Ca²⁺ oscillations evoked in HeLa cells.

Relationship between [IP₃]_c and [Ca²⁺]_c during the rising phase of Ca²⁺ spikes
Spatiotemporal profiles of both [Ca²⁺]_c and cytosolic [IP₃] at the onset of initial Ca²⁺ spikes in HeLa cells after mGluR5a stimulation were monitored with the fast acquisition equipment (see Materials and methods). Images were acquired every 246 ms. Fig. 5 shows the spatiotemporal patterns of [Ca²⁺]_c and [IP₃]_c when the first Ca²⁺ spike occurred. The rapid [Ca²⁺]_c rise occurred in HeLa cells stimulated with 100 µM glutamate with little or no pacemaker Ca²⁺ rise (Fig. 5 B). No clear initiation site of the [Ca²⁺]_c rise was detected in the cell shown (Fig. 5 A). The [IP₃]_c rise was found to precede the abrupt [Ca²⁺]_c rise (Fig. 5, B and C) in 48 of 52 cells, and [IP₃]_c gradually rose almost homogeneously in the cells stimulated (Fig. 5 A). The onset of the [IP₃]_c rise, which was identified as an increase greater than twice the SD of the baseline signals, preceded the onset of the [Ca²⁺]_c rise by 1.11 ± 1.75 s (n = 52). The rate of [IP₃]_c rise did not accelerate during the rising phase of the Ca²⁺ spike (Fig. 5, B and C, between c and e). If the regenerative IP₃ production mediated by PLC drives the rising phase of Ca²⁺ spikes, the rate of [IP₃]_c rise should peak when the rate of [Ca²⁺]_c rise is at its maximum. To test this possibility, the fluorescent signals of both Indo-1 and IRIS-1 were differentiated and aligned at the time when the rate of [Ca²⁺]_c rise reached its maximum (Fig. 5, D and E). As shown in Fig. 5 E, the rate of [IP₃]_c rise did not peak when the rate of [Ca²⁺]_c increase was at its maximum, indicating that the steep rise in [Ca²⁺]_c occurred without any acceleration of the [IP₃]_c rise. A similar relationship was observed between [Ca²⁺]_c and [IP₃]_c during the rising phases of the subsequent Ca²⁺ spikes (Fig. 6 ). As reported in histamine-stimulated HeLa cells (Bootman and Berridge, 1996), the shape of the first and the subsequent Ca²⁺ spikes was different in mGluR5a-expressing HeLa cells stimulated with glutamate. The subsequent spikes had more extended pacemaker activity, and the rate of the [Ca²⁺]_c rise after the pacemaker activity of the subsequent spikes was slower than that of the first spike (Fig. 6 A). As shown in Fig. 6, [IP₃]_c started to increase before the onset of the pacemaker [Ca²⁺]_c rise of the second, third, and fourth Ca²⁺ spikes (filled and open arrowheads), and the [IP₃]_c increase did not accelerate during the period of the rapid [Ca²⁺]_c rise of these Ca²⁺ spikes (between the thin vertical lines). The number of cells that showed [IP₃]_c rises preceding [Ca²⁺]_c increases and the mean interval between the onset of the [IP₃]_c rise and the onset of the [Ca²⁺]_c rise are summarized in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200512141/DC1). These results suggest that regenerative IP₃ production does not drive the abrupt Ca²⁺ increase of either the first or the subsequent Ca²⁺ spikes in mGluR5a-stimulated HeLa cells.

View larger version (47K): [in this window] [in a new window]   Figure 5. IP3 dynamics during the first Ca2+ spikes evoked in mGluR5a-expressing HeLa cells. (A) Pseudocolor images of Indo-1 and IRIS-1 signals in mGluR5a-expressing HeLa cells treated with 100 µM glutamate. The fluorescent signal of Venus of IRIS-1 before the glutamate stimulation is shown as a gray-scale image. Emission at 460–490 nm (F) divided by its basal level (Fbase) and ECFP/Venus emission ratio (R) divided by its basal level (Rbase) are shown for Indo-1 (F/Fbase) and IRIS-1 (R/Rbase), respectively. Images at times a–g, which are indicated in B, are shown. Bar, 20 µm. (B and C) Time courses of emission changes of Indo-1 (–F/Fbase; B) and IRIS-1 (R/Rbase; C) during the first Ca2+ spike evoked by 100 µM glutamate. Signals in the area shown in the gray-scale image (A) have been plotted. Broken horizontal lines indicate baseline levels. Broken vertical lines (c and e) indicate the onset and the end of the abrupt [Ca2+]c rise. (D and E) Differentiated signals of Indo-1 (D) and IRIS-1 (E) aligned by the time when the differentiated Indo-1 signal was at its maximum (frame 0). All data were collected from relatively small regions (40 x 40 pixels; 12.8 x 12.8 µm) located in the cytosol. Error bars correspond to the SD (n = 13).

View larger version (16K): [in this window] [in a new window]   Figure 6. IP3 dynamics during the first and the subsequent Ca2+ spikes in mGluR5a-expressing HeLa cells. Time courses of emission changes of Indo-1 (–F/Fbase; A) and IRIS-1 (R/Rbase; B) during the first four Ca2+ spikes evoked by 100 µM glutamate. Broken horizontal lines indicate baseline levels. Vertical lines indicate the time of the onset (open circle) and the end (closed circle) of the abrupt [Ca2+]c rises. Open and closed arrowheads indicate the time of onset of the increase in the Indo-1 and IRIS-1 signals, respectively. The asterisk represents an artifact.

View larger version (13K): [in this window] [in a new window]   Figure 7. Ca2+-dependent component of IP3 production in HeLa cells. Cells were treated with 1 µM thapsigargin in the absence of extracellular Ca2+ for 5 min and then exposed to 2 mM extracellular Ca2+ (A, open horizontal bar), 3 µM histamine (B, closed horizontal bar), or 3 µM histamine plus 2 mM extracellular Ca2+ (C, open and closed horizontal bars). Indo-1 signals (R/Rbase; top) and IRIS-1 signals (R/Rbase; bottom) of three cells are shown. Arrowheads point to transient [IP3]c increases. Broken horizontal lines indicate the baseline level of Indo-1 and IRIS-1 signals. Broken vertical lines indicate the time of the onset of stimulation. Images were acquired every 4 s.

	Discussion

Top Abstract Introduction Results Discussion Materials and methods References

Positive-feedback element that drives the steep rising phase of Ca²⁺ spikes
Meyer and Stryer (1988) proposed that cytosolic Ca²⁺ and IP₃ are a pair of reciprocally coupled messengers based on evidence that IP₃ induces cytosolic Ca²⁺ increase by triggering Ca²⁺ release from intracellular Ca²⁺ stores and that Ca²⁺ enhances IP₃ production by activating PLC. Their model predicts that the cooperative opening of IP₃R by the binding of multiple IP₃ molecules and positive-feedback regulation of PLC by cytosolic Ca²⁺ lead to the bistability of the system (Meyer and Stryer, 1988) and that IP₃ spikes in synchrony with Ca²⁺ spikes (we use "spike" to mean a threshold phenomenon characterized by an explosive rise and constant amplitude independent of stimulus intensity; Meyer and Stryer, 1988, 1991). We used the cytosolic IP₃ sensor IRIS-1 to directly test the cross-coupling hypothesis in living HeLa cells and found that no IP₃ spikes were detected during Ca²⁺ oscillations, according to the aforementioned definition, because the IRIS-1 signals did not exhibit an explosive rise during the rising phase of Ca²⁺ spikes (Figs. 5 and 6), and even when fluctuations were detected the absolute value of the peak level of fluctuations in IRIS-1 signals was not constant during Ca²⁺ oscillations evoked by mGluR5a stimulation (Figs. 4 and 6). The dynamic range of the IRIS-1 signals seems to be wider than the dynamic range of [IP₃]_c changes evoked by extracellular stimuli (Fig. S1 C), and the sevenfold lower IP₃ binding affinity mutant of IRIS-1, IRIS-1.2, also failed to detect IP₃ spikes (Fig. S1 E). The reaction of IRIS-1 is thought to be fast enough to detect [IP₃]_c changes during Ca²⁺ oscillations in mGluR5a-expressing HeLa cells, which exhibited 36.9 ± 7.4 mHz of Ca²⁺ oscillations (Fig. S2 D). Thus, the failure to detect IP₃ spikes was not due to inability because of either the dynamic range or time resolution of IRIS-1. Fast acquisition of both Ca²⁺ and IP₃ signals revealed that [IP₃]_c started to increase before the pacemaker rise in [Ca²⁺]_c, and the abrupt increase in [Ca²⁺]_c, which forms the rising phase of Ca²⁺ spikes, was not accompanied by a detectable acceleration in the [IP₃]_c increase (Figs. 5 and 6). The cytosolic Ca²⁺ increases actually induced IP₃ production in HeLa cells without G protein–coupled receptor activation (Fig. 7 A), but it was a relatively slow process compared with the IP₃ production evoked by histamine (Fig. 7 B). Receptor activation and [Ca²⁺]_c increases have a synergistic effect on IP₃ production, but it was not sufficiently strong to generate IP₃ spikes even when [Ca²⁺]_c was persistently elevated (Fig. 7 C). Although we cannot completely exclude the possibility that the local rapid rise in [IP₃]_c contributes to the generation of the rising phase of Ca²⁺ spikes, we used relatively small regions (Fig. 5 A), not the entire cytosol, to analyze the relationship between [Ca²⁺]_c and [IP₃]_c and found that any acceleration in the [IP₃]_c increase was not detected in the region in which [Ca²⁺]_c rose abruptly (Fig. 5, C and E). We therefore concluded that IP₃ spikes do not occur during Ca²⁺ oscillations in HeLa cells and that the positive-feedback regulation of PLC by cytosolic Ca²⁺ is not a prime mechanism for the generation of the upstroke of Ca²⁺ spikes.

What is the positive-feedback element that drives the rising phase of Ca²⁺ spikes? One plausible mechanism is Ca²⁺-induced Ca²⁺ release (CICR) mediated by IP₃R. IP₃R is activated by cytosolic Ca²⁺, and positive-feedback regulation by cytosolic Ca²⁺ enables the Ca²⁺ released by one receptor to excite its neighbors, thereby igniting a regenerative Ca²⁺ wave. However, we did not test the CICR hypothesis in the present study. The CICR hypothesis needs to be evaluated more directly under native conditions in future studies.

Mechanisms of initiation of the steep rising phase of Ca²⁺ spikes
We found that [IP₃]_c started to increase before the pacemaker rise in [Ca²⁺]_c and that the subsequent steep rise in [Ca²⁺]_c was unaccompanied by any acceleration in the [IP₃]_c increase (Figs. 5 and 6). The individual abrupt [Ca²⁺]_c rises of at least the first eight Ca²⁺ spikes were initiated at different concentrations of [IP₃]_c (Fig. 4 C), and the Ca²⁺ spikes of mGluR5a-expressing cells stimulated with 100 µM glutamate and cells stimulated with 3 µM histamine were triggered at different IP₃ concentrations (Fig. 4 C). These results indicate that a constant threshold [IP₃]_c is not involved in the initiation of the steep Ca²⁺ rise in HeLa cells. What is the actual trigger for generation of the rapid upstroke of Ca²⁺ spikes? Numerous observations have shown that elevation of [IP₃]_c to submaximal levels causes only a transient, partial release of Ca²⁺, whereas further increments of [IP₃]_c evoke an additional transient Ca²⁺ release (Muallem et al., 1989; Taylor and Potter, 1990; Bootman et al., 1992; Ferris et al., 1992; Hirota et al., 1995; Yao et al., 1995). Subsequent elevations of [IP₃]_c yielded the same degree of Ca²⁺ release as the first [IP₃]_c elevation, and the retention of responsiveness has been called "incremental detection" (Meyer and Stryer, 1990). However, because we did not detect rapid increments in [IP₃]_c when the abrupt [Ca²⁺]_c rises were triggered (Fig. 5, D and E), IP₃-sensitive Ca²⁺ stores did not respond to the rate of [IP₃]_c increase in the cells examined. A time-dependent decrease in the IP₃ binding affinity of IP₃-gated Ca²⁺ release channels and/or multiple Ca²⁺ stores with different levels of IP₃ sensitivity could account for this phenomenon. Ca²⁺ influx across plasma membranes might be involved in the initiation of repetitive spikes (Sneyd et al., 2004). Further analysis is required for the elucidation of the Ca²⁺ spike initiation mechanism.

IP₃ metabolism in living cells
The mechanism underlying the generation of Ca²⁺ oscillations has been extensively investigated using various theoretical models. What kind of uncertainty is involved in such analyses? To address this issue, we tried to reproduce the experimental measurements of both IP₃ and Ca²⁺ dynamics using a model based on previously reported models that incorporate the biphasic cytosolic Ca²⁺ dependency of IP₃R and Ca²⁺ feedback on the production of IP₃ (Meyer and Stryer, 1991; De Young and Keizer, 1992; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200512141/DC1). The model exhibited sharp Ca²⁺ oscillations accompanied by IP₃ oscillations (Fig. S3, A and B) with the original parameters (Table S2; De Young and Keizer, 1992). When we compared the calculated trace of cytosolic IP₃ with the experimental measurements (Fig. 4), the [IP₃]_c change predicted by the model was obviously too fast. We found that an 60-fold decrease in the rate of IP₃ production (Fig. S3 E and Table S2) and a 30-fold decrease in the rate of IP₃ degradation (Fig. S3 F and Table S2) produced reasonable [IP₃]_c changes (Fig. S3 C) accompanied by repetitive Ca²⁺ spikes (Fig. S3 D). The calculated Ca²⁺ dynamics (Fig. S3 D), however, were significantly different from the experimentally observed Ca²⁺ dynamics (Fig. 4) in terms of frequency and latency. Because the apparent IP₃ sensitivity for the generation of Ca²⁺ spikes progressively decreases during Ca²⁺ oscillations (Fig. 4 C), this property must be incorporated into the IP₃R model. It is clear that the actual IP₃ metabolism in living cells is slower than previously estimated (Meyer and Stryer, 1991; De Young and Keizer, 1992). A similar conclusion was obtained from the estimation of the lifetime of IP₃ in N1E-115 neuroblastoma cells (Wang et al., 1995).

Allbritton et al. (1992) proposed that Ca²⁺ is a local messenger, whereas IP₃ is a global messenger based on their diffusion constants estimated in isolated cytosol. We propose that Ca²⁺ is a fast messenger and IP₃ a slow messenger, based on the evidence that the concentration change of cytosolic IP₃ is slower than that of Ca²⁺ in living cells. IP₃R functions as a pulse generator in the signal transduction cascade that translates the information from the slow IP₃ signal to the fast Ca²⁺ signal. Elucidation of the encoding algorithm of this signal conversion is the next challenge in terms of better understanding the molecular basis of Ca²⁺ signaling.

	Materials and methods

Top Abstract Introduction Results Discussion Materials and methods References

 
Materials
An anti-GFP antibody and an anti-mGluR5 antibody were purchased from MBL International Corporation and Upstate Biotechnology, respectively.

Gene construction
The IP₃ binding core cDNA was obtained by PCR from pBact-ST-neoB-C1 (Furuichi et al., 1989; Miyawaki et al., 1990). Venus (Nagai et al., 2002) and ECFP cDNAs were fused to the 5' and the 3' end, respectively, of the IP₃ binding domain to produce IRIS proteins. Mutations of T267A and K508Q or K249Q were introduced as described elsewhere (Sawano and Miyawaki, 2000) to create an IP₃ binding–deficient mutant and a low IP₃ binding–affinity mutant, respectively. IP₃ sensor cDNAs were cloned in BamHI and XhoI sites of pcDNA3.1 zeo(+) (Invitrogen) that contains 6x Histidine tag (Fig. 1 A) in NheI to HindIII site for the expression in mammalian cells. The multicloning site of pcDNA3.1 zeo(+) was amplified by PCR and cloned into SalI site of pFastBacI (Invitrogen). IRIS-1 and IRIS-1–Dmut were digested by NheI and XhoI from pcDNA3.1 zeo(+) and inserted to the modified pFastBacI for the expression in Sf9 cells. Venus or ECFP cDNA was fused to 5' end of cDNA corresponding to amino acid residues 11–140 of rat PLC1 to create V-PHD (provided by M. Hattori, Nagoya City University, Aichi, Japan) and C-PHD, respectively. The resulting cDNAs were cloned in pcDNA3.1 zeo(+). Rat mGluR5a cDNA inserted into pME18S (Kawabata et al., 1996) was a gift from S. Nakanishi (Osaka Bioscience Institute, Osaka, Japan).

Expression, purification, and characterization in vitro
In vitro characterization of IRIS proteins were performed by using lysates prepared from COS7 or Sf9 cells expressing IRIS proteins and IRIS proteins purified from Sf9 cells. COS7 cells expressing IRIS proteins were lysed with the solution containing 10 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 0.5% NP-40. His-tagged IRIS-1 and IRIS-1–Dmut proteins were expressed by means of Sf9 baculovirus–expressing system (Invitrogen) and were purified as follows. Sf9 cells were suspended in homogenization solution containing 10 mM Hepes, pH 7.8, 100 mM NaCl, 5 mM 2-mercaptoethanol, 0.1% NP-40, and proteinase inhibitors (0.1 mM PMSF, 10 µM Leupeptin, 10 µM Pepstatin A, and 10 µM E60) and were homogenized at 1,000 rpm for 10 strokes at 4°C. Homogenates were applied to Probond resin (Invitrogen), and His-tagged proteins were eluted with 300 mM imidazole. Proteins were then dialyzed for 30 min against the solution containing 10 mM Hepes, pH 7.4, and 100 mM KCl. The dialyzed proteins were loaded to HiTrap heparin columns and eluted with the high KCl solution containing 10 mM Hepes, pH 7.4, and 250 mM KCl. Proteins were diluted by adding two volumes of the solution containing 10 mM Hepes, pH 7.4, and 100 mM KCl and concentrated with centrifugal filter devices (30,000 MWCO; Amicon Ultra 15 [Millipore]). For the evaluation of the rate of changes in IRIS-1 signals, IRIS-1–expressing Sf9 cells were suspended in 10 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.01% NP-40, and proteinase inhibitors and were homogenized at 1,000 rpm for 10 strokes at 4°C. The homogenates were centrifuged at 20,000 g for 30 min, and the supernatants were used for stopped-flow fluorescent measurements. The number of IRIS-1 molecules in the supernatants was quantified by the equilibrium IP₃ binding analysis using [³H]IP₃ as described previously (Iwai et al., 2005). IRIS signals were measured with a spectrofluorometer (FP-750; Jasco).

Imaging
IRIS cDNAs inserted in pcDNA3.1 zeo(+) were transfected into HeLa cells with transfection reagents (TransIT; Mirus). After 12–36 h, cells were used for imaging. After loading the cells with 5 µM Indo-1 AM (DOJINDO), imaging was performed under the constant flow (2 ml/min) of the balanced salt solution containing 20 mM Hepes, pH 7.4, 115 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 2 mM CaCl ₂, and 10 mM glucose as an imaging media at 37°C. Imaging was performed at 37°C through an inverted microscope (IX-71 or IX-81; Olympus) with a cooled charge-coupled device camera (ORCA-ER; Hamamatsu Photonics) and a 40x (NA 1.35) objective. A 333–348-nm excitation filter and pair of 420–440- and 460–510-nm emission filters and a 425–445-nm excitation and pair of 460–510-nm (cyan) and 525–565-nm (yellow) emission filters were used for fluorochromes (Indo-1 and IRIS, respectively). Beam-splitter mirrors (400 and 450 nm) were alternately inserted into the light path for Indo-1 and IRIS, respectively. Images were acquired at 0.25 Hz, with an exposure time of 100 or 150 ms. For the fast acquisition (4.07 Hz) of fluorescent images of IRIS-1 and Indo-1, an emission splitter (W-view; Hamamatsu Photonics) was used with a fast light source exchanger (DG-4; Sutter Instrument Co.) on the IX71 inverted microscope. Sequential excitation of IRIS-1 and Indo-1 was performed by using a 450-nm dichroic mirror and two excitation filters (a 425–445-nm filter for IRIS-1 and a 333–348-nm filter for Indo-1). Dual emission at 460–490 nm (for IRIS-1 and Indo-1) and >520 nm (for IRIS-1) split with a 460–490-nm filter, a long-path 520-nm barrier filter, and two 505-nm dichroic mirrors equipped in W-view. Images were acquired at 4.07 Hz, with an exposure time of 100 or 150 ms. Acquisition was performed with the custom software TI Workbench (written by T. Inoue). Off-line analysis was performed with TI Workbench combined with Igor Pro software (WaveMetrics). Spectral analysis of Ca²⁺ oscillations was performed as described previously (Uhlen, 2004).

Stopped-flow fluorescent measurement
The change in the intensity of Venus fluorescence (525 ± 20 nm) of IRIS-1 after the addition of various concentrations of IP₃ was monitored using the FP-750 spectrofluorometer with a stopped-flow rapid mixing accessory (RX2000; Applied Photophysics). The IRIS-1–containing supernatants (final 2.5 nM of IRIS-1) were mixed with a 24-fold–excess volume of 10 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, and 0.01% NP-40 containing various concentrations of IP₃ at 25°C, and the change of the intensity of Venus fluorescence of IRIS-1 excited at 440 ± 20 nm was monitored at 1,000 Hz. At least 10 traces were averaged and were used for the nonlinear regression analysis with Igor Pro software. The IP₃ dependency of FRET changes of IRIS-1 at equilibrium was monitored using the same batches of the supernatants as used for the kinetic measurements with a 1-cm cuvette and the FP-750 spectrofluorometer.

Online supplemental material
Fig. S1 shows effects of PLC inhibitors on emission changes of IRIS-1 elicited by 10 µM of histamine and evaluation of IRIS-1 signals observed in living HeLa cells. Fig. S2 shows the rate of reaction of IRIS-1. Fig. S3 shows simulation of IP₃ and Ca²⁺ dynamics. Table S1 shows numbers of cells that showed [IP₃]_c rises preceding [Ca²⁺]_c increases, the mean intervals between the onset of [IP₃]_c rises, and the onset of [Ca²⁺]_c rises. Table S2 shows the parameters used to calculate IP₃ and Ca²⁺ dynamics. The supplemental text shows evaluation of the effect of the rate of FRET changes of IRIS-1 upon IP₃ binding. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200512141/DC1.

	Acknowledgments

 
We thank Drs. Takeshi Nakamura, Takeharu Nagai, Masaki Sano, Toshio Aoyagi, Hideki Nakamura, Yoko Tateishi, and Sachiko Ishida for fruitful discussions and Ms. Naoko Ogawa and Miwako Iwai for excellent technical support.

This work was supported by The 21st Century Center of Excellence program, the Center for Integrated Brain Medical Science, and grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to T. Matsu-ura (18770144), T. Michikawa (17017013 and 17570128), and T. Inoue and K. Mikoshiba (15100006); the Ministry of Health, Labor and Welfare to T. Inoue; and the Moritani Scholarship Foundation to T. Michikawa.

Submitted: 27 December 2005

Accepted: 3 May 2006

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