title: Highly Sensitive Measurement of Inositol 1,4,5-Trisphosphate by Using a New Fluorescent Ligand and Ligand Binding Domain Combination.
abstract: Based on the results of our previous adenophostin A structure-activity relationship studies, two fluorescent inositol 1,4,5-trisphosphate (IP3 ) receptor ligands, fluorescent adenophostin A (FADA) and fluorescent low-affinity ligand (FLL), were designed. Binding of the fluorescent ligands to the fluorescent IP3 sensor in protein-expressing cells caused FRET. This principle was extended to a cell-free assay system by using the fluorescent IP3 sensor bound to agarose beads. The effect of FLL on the FRET signal was reduced by subsequent addition of IP3 . The IC50 values of IP3 on the FRET signals were 139.7 and 352.1 nm for 30 and 100 nm FLL, respectively. This method allowed quantitative measurement of IP3 concentrations below 10 nm and was applied to measure cytosolic IP3 concentrations in COS-7 cells and to examine the potency of synthesized adenophostin A analogues.
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Kita-12Nishi-6, Kita-ku, Sapporo, 060-0812, Japan.
authors.forename: Tai
authors.initials: T
authors.lastname: Oura
authors.affiliation: Department of Pediatric Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Kita-121757 Kanazawa, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Murata
authors.affiliation: Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Kita-121757 Kanazawa, Kita-12Ishikari-Tobetsu, Hokkaido, 061-0293, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Morita
authors.affiliation: Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Kita-121757 Kanazawa, Kita-12Ishikari-Tobetsu, Hokkaido, 061-0293, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nezu
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Kita-12Nishi-6, Kita-ku, Sapporo, 060-0812, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Arisawa
authors.affiliation: Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Kita-12Nishi-6, Kita-ku, Sapporo, 060-0812, Japan. shu@pharm.hokudai.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shuto
authors.affiliation: Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Kita-121757 Kanazawa, Kita-12Ishikari-Tobetsu, Hokkaido, 061-0293, Japan. tanimura@hoku-iryo-u.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tanimura
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis*; D008024:Ligands; D015394:Molecular Structure
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; C085029:adenophostin A; D015544:Inositol 1,4,5-Trisphosphate; D000241:Adenosine
keywords: FRET; fluorescent probes; inositol 1,4,5-trisphosphate; ligand-binding domain; sensors