GENIe: A bright new genetically‐encoded fluorescent biosensor for monitoring cGMP

Abstract

GENIe is a Genetically Encoded Nucleotide Indicator for monitoring cGMP, a second messenger in cell signaling that mediates many important physiological processes. Phosphodiesterase (PDE) inhibitors and soluble guanylate cyclase (sGC) activators modulate cGMP levels to produce beneficial effects on pulmonary and heart disease, erectile dysfunction, and other conditions. The timing, location and strength of cGMP signaling is tightly regulated by nearly 100 isoforms of cyclic nucleotide PDEs (Bender and Bravo 2006). Some isoforms are specific for cGMP, while others are specific for cAMP. Some are nonspecific and act on both cAMP and cGMP.We have developed a new, very bright mNeonGreen fluorescent biosensor to monitor cGMP levels in any living cell type. The sensor produces robust changes in fluorescence intensity that are readily detected on fluorescence imaging systems or plate readers. Since different PDE isoforms have different substrate specificities, we did parallel assays of cGMP and cAMP in HEK293 cells expressing either the cGMP biosensor (GENIe) or the cAMP biosensor (cADDis). These sensors produced markedly different fluorescent profiles of cGMP and cAMP responses when treated with different PDE inhibitors. Our results indicate that the combination of these assays provide useful insights into the specific action of a particular PDE inhibitor in a living cell. Since these sensors can be genetically targeted, and since many biologically relevant cell types express more than one PDE, these tools provide a new approach to understanding the specific action of PDE inhibitors in health and disease.We tested GENIe in HEK293T cells on a BioTek Synergy MX automated fluorescence plate reader. cGMP production was initiated by adding sodium nitroprusside (SNP), a nitric oxide donor, in both the presence and absence of the PDE5 selective inhibitor, Sildenafil. Fluorescence intensity changed dramatically in the presence of Sildenafil, as degradation of cGMP is inhibited by the drug. To optimize the response in HEK293T cells, we over‐expressed sGC, treated with SNP, and observed a significant improvement in the amplitude of the signal accompanied by faster kinetics and an increased sensitivity to the nitric oxide donor SNP. We expect that endogenous levels of sGC will vary in different cell types and these parameters should be varied accordingly.To examine PDE inhibitor specificity, we monitored responses from both cADDis and GENIe in the presence of different PDE inhibitors. IBMX, a non‐specific inhibitor, produced similar responses from both sensors. Sildenafil and Rolipram indicated cGMP vs cAMP specificity. Sildenafil exhibited selective inhibition of the cGMP‐specific PDE5, while Rolipram exhibited selective inhibition of the cAMP specific PDE4.Support or Funding InformationFunding from NSF SBIR Phase IIB 1430878This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.