(7,8). On the other hand, oscillatory changes in [IP 3 ] i have been suggested by the observed cyclical translocation of a GFPtagged pleckstrin homology domain of PLC-␦ (GFP-PHD) (9, 10). However, in other experiments using more specific IP 3 biosensors, IP 3 was shown to accumulate gradually with little or no fluctuation during Ca 2ϩ oscillations (11). These discrepant observations may be attributable to differences between various IP 3 biosensors and a lack of quantitation.There are two types of IP 3 biosensors, GFP-PHD and IP 3 Rbased FRET sensors. GFP-PHD binds to both PIP 2 and IP 3 ; thus it has been thought that changes in [IP 3 ] i could be monitored indirectly by the release of membrane-bound GFP-PHD (9). IP 3 R-based FRET biosensors consist of the ligand-binding domain of IP 3 R and a pair of fluorescent proteins, cyan fluorescent protein and yellow fluorescent protein. Since the successful monitoring of IP 3 with LIBRA (12), the first IP 3 R-based FRET biosensor, several different groups have used similar biosensors for IP 3 monitoring (11,13,14). In principle, quantitative measurements of [IP 3 ] i are not possible with GFP-PHD. In addition, it is recognized that GFP-PHD may be released from the plasma membrane by decreases in available PIP 2 (15), which could be attributed to PIP 2 hydrolysis or the occupation by other molecules. IP 3 R-based FRET biosensors offer significant benefits for monitoring IP 3 based on their high selectivity for IP 3 and ratiometric measurement.In this study, we developed a series of improved IP 3 biosensors that exhibit high pH stability and varying IP 3 affinities. They also possess higher selectively and afford a larger dynamic range than that of original LIBRA. In combination with these * This work was supported by Grant-in-aid for Scientific Research 16390532 (to A. T.), by HAITEKU (2007) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Japan Science and Technology Agency. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

) (6).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 IP 3 -binding domain of the rat type 3 IP 3 R (amino acids 1-604) was amplified by PCR using pCB-EGFP: IP 3 R3 (7) 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. LIBRA⌬N was constructed in the same way using amino acids 227-604 of the rat type 3 IP 3 R (8). 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 LIBRA⌬N. 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 g/ml streptomycin. Cells were transfected with plasmids using LipofectAMINE

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We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca(2+) release and entry using a mutant DT40 cell line (IP(3)R-KO) in which all three IP(3)R genes had been disrupted. GFP-IP(3)R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP(3)R3 apparently co-localized with the plasma membrane. The application of IP(3) to permeabilized WT (wild-type) DT40 cells induced Ca(2+) release from internal stores. Although this did not occur in IP(3)R-KO cells it was restored by expression of GFP-IP(3)R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca(2+) response in IP(3)R-KO cells, whereas these treatments induced oscillatory or transient Ca(2+) responses in GFP-IP(3)R3-expressing IP(3)R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca(2+) entry in WT and GFP-IP(3)R3-expressing IP(3)R-KO cells but not in IP(3)R-KO cells. This BCR-mediated Ca(2+) entry was observed in the presence of La(3+), which blocks capacitative Ca(2+) entry. Thapsigargin depleted Ca(2+) stores and led to Ca(2+) entry in IP(3)R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca(2+) entry was completely blocked by La(3+), suggesting that the BCR-mediated Ca(2+) entry pathway is distinct from the capacitative Ca(2+) entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP(3)R in both IP(3)-induced Ca(2+) release and BCR-mediated Ca(2+) entry.

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