2019
DOI: 10.1038/s42003-019-0641-x
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FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons

Abstract: Several FRET (fluorescence resonance energy transfer)-based biosensors for intracellular detection of cyclic nucleotides have been designed in the past decade. However, few such biosensors are available for cGMP, and even fewer that detect low nanomolar cGMP concentrations. Our aim was to develop a FRET-based cGMP biosensor with high affinity for cGMP as a tool for intracellular signaling studies. We used the carboxyl-terminal cyclic nucleotide binding domain of Plasmodium falciparum cGMP-dependent protein kin… Show more

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Cited by 36 publications
(34 citation statements)
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“…However, as we have shown, changing the ECFP/EYFP pair to Rluc8/Venus did not have the same effect on selectivity. Additionally, the recently developed FRET-based cGMP sensor Pf PKG, which was based on PKG (rather than PDE5) from Plasmodium falciparum, showed a high cGMP affinity of about 23 nM, but this was coupled with a high cAMP affinity of about 4.6 µM, suggesting that it may be difficult to decouple the affinities of the two cyclic nucleotides for cGMP-binding domains [33]. In fact, the authors even mutated the cGMP-binding domain from PKG in an attempt to reduce the affinity of cAMP, but were unsuccessful [33].…”
Section: Discussionmentioning
confidence: 99%
“…However, as we have shown, changing the ECFP/EYFP pair to Rluc8/Venus did not have the same effect on selectivity. Additionally, the recently developed FRET-based cGMP sensor Pf PKG, which was based on PKG (rather than PDE5) from Plasmodium falciparum, showed a high cGMP affinity of about 23 nM, but this was coupled with a high cAMP affinity of about 4.6 µM, suggesting that it may be difficult to decouple the affinities of the two cyclic nucleotides for cGMP-binding domains [33]. In fact, the authors even mutated the cGMP-binding domain from PKG in an attempt to reduce the affinity of cAMP, but were unsuccessful [33].…”
Section: Discussionmentioning
confidence: 99%
“…These biosensors (termed PfPKG) were produced by flanking the cyclic nucleotide binding domain of Plasmodium falciparum cGKI with different FRET pairs. Experiments in rat cardiomyocytes and stellate ganglion neurons revealed that the PfPKG biosensors display high affinity for cGMP (Calamera et al, 2019). Hence, they are likely to serve as new tools for real-time measurement of low intracellular cGMP concentrations in living cells.…”
Section: New Developments In Cgmp-based Treatment Approaches and Thermentioning
confidence: 99%
“…The development of genetically encoded sensors based on Föster resonance energy transfer (FRET) effect is a well-established methodology for the non-invasive and real-time study of a plethora of cellular events Berrera et al, 2008;Meng and Sachs, 2011;Calamera et al, 2019).…”
Section: Introductionmentioning
confidence: 99%
“…So far, the sensor with higher affinity for cGMP has an EC50. ∼ 40 nM (Niino et al, 2009;Calamera et al, 2019).…”
Section: Introductionmentioning
confidence: 99%