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

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.

In vivo model with targeted cAMP biosensor reveals changes in receptor–microdomain communication in cardiac disease

“…However, since PLN forms pentamers that may rearrange upon PKA-mediated phosphorylation, we sought to study whether these processes may affect FRET responses measured by the Epac1-PLN sensor. To do so, we introduced the previously characterized R279E mutation, which renders the sensor insensitive to cAMP 18 . Importantly, this mutant construct did not show any change in FRET upon b-adrenergic stimulation ( Supplementary Fig.…”

“…8), suggesting that the actual differences between b 1 -AR responses in the SERCA2a microdomain and the cytosol might be even bigger. Using the in vitro calibration curves and our previously described protocol 18 , we converted FRET ratio values into the absolute cAMP concentrations and found about fourfold higher stimulated cAMP values in the vicinity of SERCA2a as compared with the bulk cytosol (3.9±0.4 mM in the cytosol versus 15.3±2.8 mM at SERCA2a, respectively, means ± s.e., Po0.01 by one-way analysis of variance (ANOVA)). Pre-treatment of cells with the unselective PDE inhibitor 3-isobutyl-1-methylxanthin (IBMX) blunted this difference in FRET responses ( Fig.…”

“…Therefore, this represents an important limitation of our study. However, a reliable strategy to overcome this problem is to perform accurate calibration of both sensors and to recalculate the FRET ratio data into absolute cAMP concentrations or equivalents of the generalized forskolin responses 18,36 . We chose the first option and provided the accurately calculated cAMP values for all key experiments, which directly compare responses from both sensors to support our conclusions (see Figs 2e and 4e).…”

“…The resulting construct was subcloned via KpnI/XhoI restriction sites into a vector containing the a-MHC promoter, which was used for generation of TG mice 28 . In addition to the functional Epac1-PLN construct, the cAMP-insensitive mutant was generated by exchanging the cAMP-binding domain sequence in this sensor with a sequence containing the R279E point mutation 18 . To generate dark CFP and dark YFP constructs, the following mutations were introduced into Epac1-PLN sequence via site-directed mutagenesis: W66G in CFP, and Y66G in YFP.…”

Cigarette smoke up‐regulates PDE3 and PDE4 to decrease cAMP in airway cells

“…(2011), with minor modifications (Sprenger et al ., 2015). A lung slice was placed in the Attofluor cell chamber (Invitrogen, Landsmeer, Netherlands).…”