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

Abstract

) (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

Identification and Functional Characterization of Voltage-dependent Calcium Channels in T Lymphocytes

“…Initially, investigators suggested that a plasma membrane IP 3 R Ca 2ϩ channel, similar to the IP 3 R found in the ER, was responsible for Ca 2ϩ influx in T lymphocytes (9). A recent study confirmed that T lymphocytes express three isoforms of the IP 3 R Ca 2ϩ channel as integral plasma membrane proteins (10). However, the IP 3 R isoforms exhibit functional redundancy; defining the respective contributions of these channels to Ca 2ϩ influx during T cell activation has therefore been difficult (11).…”

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

“…Recent developments, however, have seen the arrival of novel fluorescent probes to study intracellular IP 3 dynamics. For example, a biosensor derived from the IP 3 binding domain of type-3 IP 3 R, termed 'LIBRA', was employed to measure IP 3 concentrations in living cells [38]. Similarly, the intracellular translocation of a pleckstrin homology domain from phospholipase C ∂1 fused to GFP was used to estimate [IP 3 ] [39], and to measure agonist-induced oscillatory changes of IP 3 concentration [40].…”

Regulation of Inositol 1,4,5-Trisphosphate Receptor-mediated Calcium Release by the Na/K-ATPase in Cultured Renal Epithelial Cells

“…Biotinylation of Cell Surface Proteins-The cells were washed twice with ice-cold phosphate-buffered saline and then biotinylated as described previously (3,22). The biotinylated cells were then lysed with 1 ml of radioimmunoprecipitation buffer for 30 min at 4°C.…”