A novel fluorescent method employing the FRET-based biosensor “LIBRA” for the identification of ligands of the inositol 1,4,5-trisphosphate receptors |
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Authors: | Akihiro Nezu Akihiko TanimuraTakao Morita Akiko ShitaraYosuke Tojyo |
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Affiliation: | Department of Dental Pharmacology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan |
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Abstract: | LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP3) and is composed of the ligand-binding domain of the rat type 3 IP3 receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP3-insensitive mutant LIBRA-N to compounds known to inhibit IP3-induced Ca2+ release. Heparin, a competitive antagonist of IP3 receptors, increased the emission ratio of LIBRA but not that of LIBRA-N. In contrast, 2-aminoethoxydiphenyl borate, a known non-competitive inhibitor of IP3 receptor, decreased the emission ratios of both LIBRA and LIBRA-N. Thus, the concurrent use of LIBRA-N with LIBRA identifies nonspecific responses. These results indicate that LIBRA and its mutant control can be used to detect specific agonists and antagonists of IP3 receptors. We also demonstrate the utility of LIBRA and LIBRA-N in discriminating between specific and nonspecific responses in intact cells. |
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Keywords: | 2APB, 2-aminoethoxydiphenyl borate Ach, acetylcholine CCD, charge-coupled device CFP, cyan fluorescent protein ECFP, enhanced CFP EYFP, enhanced YFP FRET, fluorescence resonance energy transfer ICM, intracellular-like medium IP3, inositol 1,4,5-trisphosphate IP3R, IP3 receptor PLC, phospholipase C YFP, yellow fluorescent protein |
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