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).