Quantitative Measurement of cAMP Concentration Using an Exchange Protein Directly Activated by a cAMP-Based FRET-Sensor

Förster resonance energy transfer (FRET)-based biosensors for the quantitative analysis of intracellular signaling, including sensors for monitoring cyclic adenosine monophosphate (cAMP), are of increasing interest. The measurement of the donor/acceptor emission ratio in tandem biosensors excited at the donor excitation wavelength is a commonly used technique. A general problem, however, is that this ratio varies not only with the changes in cAMP concentration but also with the changes of the ionic environment or other factors affecting the folding probability of the fluorophores. Here, we use a spectral FRET analysis on the basis of two excitation wavelengths to obtain a reliable measure of the absolute cAMP concentrations with high temporal and spatial resolution by using an “exchange protein directly activated by cAMP”. In this approach, FRET analysis is simplified and does not require additional calibration routines. The change in FRET efficiency (E) of the biosensor caused by [cAMP] changes was determined as ΔE = 15%, whereas E varies between 35% at low and 20% at high [cAMP], allowing quantitative measurement of cAMP concentration in the range from 150 nM to 15 μM. The method described is also suitable for other FRET-based biosensors with a 1:1 donor/acceptor stoichiometry. As a proof of principle, we measured the specially resolved cAMP concentration within living cells and determined the dynamic changes of cAMP levels after stimulation of the Gs-coupled serotonin receptor subtype 7 (5-HT7)

An Ion-insensitive cAMP Biosensor for Long Term Quantitative Ratiometric Fluorescence Resonance Energy Transfer (FRET) Measurements under Variable Physiological Conditions*

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-HT1A receptor