title: An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions.
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.
authors: Department of Neuro- and Sensory Physiology, University of Göttingen, 37073 Göttingen, Germany. psaloni@gwdg.de
authors: Petrus S
authors: PS
authors: Salonikidis
authors: Marcus
authors: M
authors: Niebert
authors: Tim
authors: T
authors: Ullrich
authors: Guobin
authors: G
authors: Bao
authors: Andre
authors: A
authors: Zeug
authors: Diethelm W
authors: DW
authors: Richter
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000032:analysis / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D020662:Guanine Nucleotide Exchange Factors / Q000096:biosynthesis; D051379:Mice; D044282:Receptor, Serotonin, 5-HT1A / Q000378:metabolism
chemical_list: C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D044282:Receptor, Serotonin, 5-HT1A; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D002118:Calcium