ArticlePloS one2015
Measurement of inositol 1,4,5-trisphosphate in living cells using an improved set of resonance energy transfer-based biosensors.
Article in PloS one, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 26 citations in OpenAlex.
- Dynamic assessment of PI4P metabolism using split GFP based sensors.Scientific reports · 2025Article
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- Quantitative Monitoring of GPCR-Mediated Spatiotemporal IPMethods in molecular biology (Clifton, N.J.) · 2024Article
- Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- GPCRs in Intracellular Compartments: New Targets for Drug Discovery.Biomolecules · 2022Review
- BRET-Based Biosensors to Measure Agonist Efficacies in Histamine HInternational journal of molecular sciences · 2022Article
- Methods to study phosphoinositide regulation of ion channels.Methods in enzymology · 2021Article
- β-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.Cell · 2020Article
- ORP3 phosphorylation regulates phosphatidylinositol 4-phosphate and CaJournal of cell science · 2020Article
- The Role of β-Arrestin Proteins in Organization of Signaling and Regulation of the AT1 Angiotensin Receptor.Frontiers in endocrinology · 2019Review
- Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks.Chemical reviews · 2018Review
- High expression of type I inositol 1,4,5-trisphosphate receptor in the kidney of rats with hepatorenal syndrome.World journal of gastroenterology · 2018Article
- Heterologous phosphorylation-induced formation of a stability lock permits regulation of inactive receptors by β-arrestins.The Journal of biological chemistry · 2018Article
- Prostaglandin EMolecular pharmacology · 2017Article
- BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation.Biochimica et biophysica acta · 2016Article
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Authors and funding
7 authors at 3 institutions in 2 countries.
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Abstract
Improved versions of inositol-1,4,5-trisphosphate (InsP3) sensors were created to follow intracellular InsP3 changes in single living cells and in cell populations. Similar to previous InsP3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP3 receptor (InsP3R-LBD), but contain a mutation of either R265K or R269K to lower their InsP3 binding affinity. Tagging the InsP3R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonist-induced increase in InsP3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP3 and Ca2+ levels in BRET experiments, the Cameleon D3 Ca2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P2 resulted in the fall of InsP3 level, followed by the decrease of the Ca2+-signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca2+-signal preceded the fall of InsP3 level indicating an InsP3-, independent, direct regulation of capacitative Ca2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP3 sensor can be used to monitor both the increase and decrease of InsP3 levels in live cells suitable for high-throughput BRET applications.
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