title: A fluorescence-based method for evaluating inositol 1,4,5-trisphosphate receptor ligands: determination of subtype selectivity and partial agonist effects.
abstract: Inositol 1,4,5-trisphosphate (IP₃) receptors consist of three subtypes: IP₃R1, IP₃R2, and IP₃R3. Although numerous IP₃ receptor ligands have been synthesized, none of the subtype-selective ligands are known. We have developed a simple fluorescence method to examine the subtype selectivity of IP₃ receptor ligands using FRET-based IP₃ biosensors LIBRAvI, LIBRAvII, and LIBRAvIII. The addition of IP₃ or adenophostin A (ADA) to permeabilized biosensor-expressing cells increased the fluorescence ratios of these biosensors in a concentration-dependent manner, and the potency of ADA relative to that of IP₃ in terms of the changes in the fluorescence ratios of LIBRAvI, LIBRAvII, and LIBRAvIII was 43-, 22-, and 28-fold, respectively. This fluorescence-based method further showed that several ADA analogs had significant differences with respect to subtype selectivity and potency. These results highlight the important role played by the O-glycosidic structure of ADA in the selectivity of the ligands for IP₃R1, as evidenced by the modified selectivity following replacement of the 5'-hydroxyl with a phenyl or phenethyl group. We also found that one ADA analog 5'-deoxy-5'-phenyladenophostin A possessed a partial agonistic effect on IP₃R1. Together, the novel fluorescent methods described herein are useful for the evaluation of properties of IP₃R ligands, including potency, efficacy, and subtype selectivity.
authors.affiliation: Department of Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu 061-0293, Japan. tanimura@hoku-iryo-u.ac.jp
authors.forename: Akihiko
authors.initials: A
authors.lastname: Tanimura
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mochizuki
authors.forename: Takao
authors.initials: T
authors.lastname: Morita
authors.forename: Akihiro
authors.initials: A
authors.lastname: Nezu
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Tojyo
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Arisawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shuto
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives; D015374:Biosensing Techniques / Q000379:methods*; D002120:Calcium Channel Agonists / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000819:agonists / Q000378:metabolism*; D008024:Ligands
chemical_list: C515437:5'-deoxy-5'-phenyladenophostin A; D002120:Calcium Channel Agonists; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008024:Ligands; D000241:Adenosine
keywords: ADA; Adenophostin A; ECFP; EYFP; FRET; Fluorescence resonance energy transfer (FRET); Fluorescent IP(3) biosensor; HBSS-H; Hanks’ balanced salt solution buffered with Hepes; ICM; IP(3); IP(3)R; Inositol 1,4,5-trisphosphate (IP(3)); LBD; Partial agonist; Subtype selectivity; adenophostin A; enhanced cyan fluorescent protein; enhanced yellow fluorescent protein; fluorescence resonance energy transfer; inositol 1,4,5-trisphosphate; inositol 1,4,5-trisphosphate receptor; intracellular-like medium; ligand-binding domain of IP(3)R