Real‐Time FRET‐Based Measurements of Ca2+/PKA Dynamics in Plasma Membrane Microdomains of Primary Hippocampal Neurons

Abstract Both Ca 2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca 2+ /PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor‐acceptor emission and red fluorescence signal. In this detailed step‐by‐step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non‐raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real‐time measurements. Finally, we provide examples of the observed Ca 2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross‐talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.