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 kinase (PKG) flanked by different FRET pairs to generate two cGMP biosensors (Yellow PfPKG and Red PfPKG). Here, we report that these cGMP biosensors display high affinity for cGMP (EC50 of 23 ± 3 nM) and detect cGMP produced through soluble guanylyl cyclase and guanylyl cyclase A in stellate ganglion neurons and guanylyl cyclase B in cardiomyocytes. These biosensors are therefore optimal tools for real-time measurements of low concentrations of cGMP in living cells.
Cell signalling, Cellular imaging, Fluorescent proteins, Membrane proteins, Animals, Biosensing Techniques, Computer Systems, Cyclic GMP, Cyclic GMP-Dependent Protein Kinases, Fluorescence Resonance Energy Transfer, Guanylate Cyclase, HEK293 Cells, Humans, Male, Models, Molecular, Myocytes, Cardiac, Neurons, Plasmodium falciparum, Protozoan Proteins, Rats, Wistar, Recombinant Fusion Proteins, Single-Cell Analysis, Soluble Guanylyl Cyclase