FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons

摘要

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. Gaia Calamera et al. develop two cGMP biosensors, Yellow PfPKG and Red PfPKG, which bind to cGMP with high affinity. These biosensors are made of a cyclic nucleotide binding domain flanked by different FRET pairs, enabling real-time measurement of cGMP in living cells with inherently low cGMP concentrations.