Red-Shifted Epac-Based FRET cAMP Sensors for All-Optical cAMP Control and Multiparameter Imaging

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger downstream of G protein-coupled receptors (GPCRs) and a central regulator of cellular signaling. Genetically encoded exchange proteins directly activated by cAMP (Epac)-based Förster resonance energy transfer (FRET) biosensors enable real-time monitoring of cAMP dynamics in living cells, but commonly used cyan/yellow FRET pairs require short-wavelength excitation, limiting compatibility with multiplex imaging and blue-light optogenetic tools such as bacterial photoactivated adenylyl cyclases (bPACs). Here, we engineered and systematically characterized four red-shifted Epac-based single-chain FRET cAMP sensors combining yellow or orange FRET donors with red fluorescent FRET acceptors. Using ratiometric live-cell imaging, we quantified stimulus-evoked FRET responses and identified Epac_red4 as the best-performing variant, showing an approximately 55% decrease in normalized FRET after forskolin stimulation. Epac_red4 also reliably detected G_i/o-mediated decreases in cAMP following μ-opioid receptor activation. Brief 405 nm light pulses induced graded and reversible cAMP elevations using the low dark-activity variant bPAC-F198Y. Furthermore, Epac_red4 enabled analysis of cAMP recovery kinetics during phosphodiesterase inhibition and multiplex imaging of cAMP and intracellular Ca²⁺ using Fura-2 with minimal spectral and pH-related interference under physiological imaging conditions. Together, Epac_red4 represents a robust red-shifted cAMP sensor for optogenetic and multiplex signaling studies.