CaMK2rep: A Highly Sensitive Genetically Encoded Biosensor for Monitoring CaMKII Activity in Mammalian Cells

Accurately monitoring calcium/calmodulin-dependent protein kinase II (CaMKII) activity in cells remains a significant challenge due to the limited sensitivity and narrow dynamic range of existing genetically encoded sensors. Here, we introduce CaMK2rep, a novel phosphorylation-based biosensor that enables robust, specific, and high-sensitivity detection of CaMKII activity. CaMK2rep is designed with two tandem CaMKII consensus sites embedded within the native sequence context of synapsin, and its phosphorylation is detected via a phospho-specific antibody, allowing both biochemical and morphological analyses. We validated CaMK2rep in HeLa cells and cultured hippocampal neurons, demonstrating a near-linear response to CaMKII expression levels and to stimulation intensity, and no detectable cytotoxicity. To complement CaMK2rep measurements, we employed the live-cell CaMKAR (Reyes Gaido, O. E. et al. Sci. Transl. Med. 15, eabq7839 (2023)) reporter to monitor CaMKII activity dynamics. Using both tools, we investigated the role of neurogranin (Ng), a major postsynaptic calmodulin (CaM) binding protein, and obtained consistent evidence supporting a CaM-buffering model in which Ng limits basal CaMKII activation by sequestering CaM. These findings establish CaMK2rep as a sensitive, specific, and versatile biosensor for CaMKII signaling, particularly well-suited for immunoblot-based population analyses. They also illustrate the value of combining orthogonal genetically encoded tools to interrogate complex signaling mechanisms in both physiological and pathological contexts.