Fluorescent Biosensor for Quantitative Real-time Measurements of Inositol 1,4,5-Trisphosphate in Single Living Cells
| Autor: | Yosuke Tojyo, Akihiro Nezu, Akihiko Tanimura, R. James Turner, Takao Morita |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Yellow fluorescent protein
Time Factors Recombinant Fusion Proteins Green Fluorescent Proteins Molecular Sequence Data Biosensing Techniques Inositol 1 4 5-Trisphosphate Biology Transfection Biochemistry chemistry.chemical_compound Adenosine Triphosphate Bacterial Proteins Fluorescence Resonance Energy Transfer Animals Receptors Cholinergic Inositol Amino Acid Sequence Molecular Biology Chelating Agents Ionomycin Cell Biology Hydrogen-Ion Concentration Receptors Muscarinic Fluorescence Protein Structure Tertiary Rats Luminescent Proteins Spectrometry Fluorescence Förster resonance energy transfer chemistry Second messenger system Biophysics biology.protein Calcium Signal transduction Biosensor Intracellular Plasmids Protein Binding Signal Transduction |
| Zdroj: | Journal of Biological Chemistry. 279:38095-38098 |
| ISSN: | 0021-9258 |
| Popis: | The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time. LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP(3) and unaffected by concentrations of Ca(2+) and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation. |
| Databáze: | OpenAIRE |
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