ArticleThe Journal of biological chemistry2011
An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions.
Article in The Journal of biological chemistry, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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19 citing papers in PubMed, 65 citations in OpenAlex.
- Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals.Chemical reviews · 2024Review
- The cell adhesion molecule CD44 acts as a modulator of 5-HT7 receptor functions.Cell communication and signaling : CCS · 2024Article
- Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.Cell communication and signaling : CCS · 2024Article
- Genetically encoded sensors towards imaging cAMP and PKA activity in vivo.Journal of neuroscience methods · 2021Review
- Review
- A Novel Genetically Encoded Single Use Sensory Cellular Test System Measures Bicarbonate Concentration Changes in Living Cells.Sensors (Basel, Switzerland) · 2020Article
- Attenuated palmitoylation of serotonin receptor 5-HT1A affects receptor function and contributes to depression-like behaviors.Nature communications · 2019Article
- Genetically Encoded Fluorescent Biosensors Illuminate the Spatiotemporal Regulation of Signaling Networks.Chemical reviews · 2018Review
- Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development.Physiological reviews · 2018Review
- Chimeric peptide EP45 as a dual agonist at GLP-1 and NPY2R receptors.Scientific reports · 2018Article
- The role of Epac in the heart.Cellular and molecular life sciences : CMLS · 2017Review
- Genetically-encoded tools for cAMP probing and modulation in living systems.Frontiers in pharmacology · 2015Review
- Article
- Targeted activation of conventional and novel protein kinases C through differential translocation patterns.Molecular and cellular biology · 2014Article
- Coarse architecture of the transient receptor potential vanilloid 1 (TRPV1) ion channel determined by fluorescence resonance energy transfer.The Journal of biological chemistry · 2013Article
- Biophysical techniques for detection of cAMP and cGMP in living cells.International journal of molecular sciences · 2013Review
- Visualizing and manipulating focal adhesion kinase regulation in live cells.The Journal of biological chemistry · 2013Article
- Fluorescence resonance energy transfer microscopy as demonstrated by measuring the activation of the serine/threonine kinase Akt.Nature protocols · 2013Article
- An improved targeted cAMP sensor to study the regulation of adenylyl cyclase 8 by Ca2+ entry through voltage-gated channels.PloS one · 2013Article
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6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.