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FlincGs: novel, non-FRET cGMP biosensors with nanomolar sensitivity for NO-induced signaling

Previously, our lab has developed FRET-based cGMP-indicators (cygnets) to study the spatial and temporal dynamics of intracellular cGMP [1]. Cygnets have been proven to advance our understanding of NO/cGMP signaling in vascular smooth muscle cells [2]. However, FRET based indicators suffer from intrinsic technical difficulties: they require a dual emission detection system, show overall low cyan/yellow emission ratio changes, and are generally insensitive to physiological (low nanomolar) stimuli of NO. These restrictions prompted us to develop several novel, non-FRET based cGMP biosensors (FlincG:fl uorescence in dicator of c yclic G MP), which are composed of a single circular permutated EGFP (cpEGFP) fused to regulatory fragments of cGMP-dependent protein kinase (PKG). Based on the different PKG type I isoforms, we designed α-FlincG and β-FlincG. A third construct (δ-FlincG), had the entire N-terminal region truncated. All three indicators were expressed and purified from E. coli and showed cGMP selective changes in total 510 nm emission intensities of 50–200% with apparent KD,cGMP values ranging from 40 nM to 1.2 μM. Furthermore, we observed a >1000 fold selectivity for cGMP over cAMP. Interestingly, in adenovirus transfected vascular smooth muscle cells (P0), FlincG indicators detected cGMP responses to sub-nanomolar NO. The apparent EC50 values were determined as 0.3 nM, 12 nM and 7 nM for α-FlincG, β-FlincG and δ-FlincG, respectively. Due to their superior kinetic characteristics, FlincG biosensors may serve as ideal tools to elucidate cGMP signaling in cultured smooth muscle cells and in intact arteries using conventional epi-fluorescence microscopy as well as confocal fluorescence microscopy.

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Correspondence to Lydia WM Nausch.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Nausch, L.W., Dostmann, W.R. FlincGs: novel, non-FRET cGMP biosensors with nanomolar sensitivity for NO-induced signaling. BMC Pharmacol 7, S22 (2007). https://doi.org/10.1186/1471-2210-7-S1-S22

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Keywords

  • Vascular Smooth Muscle Cell
  • Confocal Fluorescence Microscopy
  • Emission Ratio
  • Selective Change
  • Emission Detection