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).

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 (×10⁻³).

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 (F_base) and ECFP/Venus emission ratio (R) divided by its basal level (R_base) are shown for Indo-1 (F/F_base) and IRIS-1 (R/R_base), 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/F_base; B) and IRIS-1 (R/R_base; 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 × 40 pixels; 12.8 × 12.8 μm) located in the cytosol. Error bars correspond to the SD (n = 13).

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 (ΔR/R_base; top) and IRIS-1 signals (ΔR/R_base; 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.

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 ΔR was defined as R − R_base, where R_base 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 (×10⁻³).

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.

IP₃ dynamics during the first and the subsequent Ca²⁺ spikes in mGluR5a-expressing HeLa cells. Time courses of emission changes of Indo-1 (−F/F_base; A) and IRIS-1 (R/R_base; 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.