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FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells

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Abstract

Real-time measurements of second messengers in living cells, such as cAMP, are usually performed by ratiometric fluorescence resonance energy transfer (FRET) imaging. However, correct calibration of FRET ratios, accurate calculations of absolute cAMP levels and actual permeabilities of different cAMP analogs have been challenging. Here we present a protocol that allows precise measurements of cAMP concentrations and kinetics by expressing FRET-based cAMP sensors in cells and modulating them with an inhibitor of adenylyl cyclase activity and a cell-permeable cAMP analog that fully inhibits and activates the sensors, respectively. Using this protocol, we observed different basal cAMP levels in primary mouse cardiomyocytes, thyroid cells and in 293A cells. The protocol can be generally applied for calibration of second messenger or metabolite concentrations measured by FRET, and for studying kinetics and pharmacological properties of their membrane-permeable analogs. The complete procedure, including cell preparation and FRET measurements, takes 3–6 d.

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Figure 1: Schematic structure of the cAMP sensor Epac1-camps and equipment setup for FRET imaging.
Figure 2: Measurements of basal cAMP concentrations in various cell types.
Figure 3: Examples of experiments showing measurements of ligand-induced changes in intracellular cAMP concentrations, as well as in kinetics and permeabilities for cAMP analogs.

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Acknowledgements

The work in authors' laboratories is supported by the Deutsche Forschungsgemeinschaft (grants SFB487 and SFB688 to M.J.L.; grant NI 1301/1-1 to V.O.N.), European Research Council (to M.J.L.) and University of Göttingen Medical Center ('pro futura' grant to V.O.N.). We thank H.-G. Genieser for critical reading of the manuscript.

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Authors and Affiliations

  1. Institute of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany

    Sebastian Börner, Angela Schlipp, Filip Berisha, Davide Calebiro, Martin J Lohse & Viacheslav O Nikolaev

  2. Rudolf Virchow Center, Deutsche Forschungsgemeinschaft (DFG) Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany

    Sebastian Börner, Angela Schlipp, Filip Berisha, Davide Calebiro, Martin J Lohse & Viacheslav O Nikolaev

  3. BIOLOG Life Science Institute, Bremen, Germany

    Frank Schwede

  4. Department of Cardiology and Pneumology, Emmy Noether Group of the DFG, Georg August University Medical Center, Göttingen, Germany

    Viacheslav O Nikolaev

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  1. Sebastian Börner
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  2. Frank Schwede
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Contributions

S.B., F.S., F.B. and V.O.N. performed experiments and analyzed the data. A.S. and D.C. prepared primary cardiomyocytes and thyroid cells. F.S., M.J.L. and V.O.N. developed the protocol. S.B. and V.O.N. wrote the paper.

Corresponding author

Correspondence to Viacheslav O Nikolaev.

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Competing interests

F.S. is an employee and a shareholder of the Biolog Life Science Institute, a company that sells chemicals including cAMP analogs.

Supplementary information

Supplementary Figure 1

Experiments aimed to determine the concentration of MDL-12,330A which is capable of fully inhibiting adenylyl cyclase activity in cells and which can be used to reliably measure Rmin values. (PDF 56 kb)

(a) Concentration-response dependence of MDL-12,330A effect on the basal FRET ratio in cardiomyocytes shows that 100 µM of this compound can fully inhibit the sensor, and application of higher inhibitor concentrations does not lead to lower Rmin values (means ± SEM, n=7), which was also true for thyroid cells (not shown). (b) In cells with low basal adenylyl cyclase activity, inhibitory effect of MDL-12,330A can be tested after stimulation of cAMP production, as shown here for 293A cells treated with the _-adrenergic receptor agonist isoproterenol. 100 µM of MDL-12,330A were also sufficient to fully block the stimulated adenylyl cyclase activity in these cells. A grey trace shows a control experiment where the buffer A was added instead of MDL-12,330A, which did not result in a rapid decrease of signal. Representative experiments, n=5.

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Börner, S., Schwede, F., Schlipp, A. et al. FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells. Nat Protoc 6, 427–438 (2011). https://doi.org/10.1038/nprot.2010.198

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