Calcium nanodomains, defined by the tight coupling of Ca 2+ sources and sensors within 20–50 nm, critically shape neuronal excitability and synaptic plasticity. However, tools to resolve these highly localized signals remain limited in both spatial precision and dynamic range. Large-conductance Ca 2+ - and voltage-activated K + (BK) channels are intrinsic components of many nanodomains, making them promising scaffolds for the development of targeted calcium sensors. Here, we report two complementary strategies for engineering BK-based fluorescent sensors. First, circularly permuted HaloTag (cpHalo) was inserted at multiple BK channel sites predicted to undergo conformational changes upon Ca 2+ binding. Structural modeling and experimental validation confirmed stable expression and trafficking to the plasma membrane. Among the variants, BK965cpHalo exhibited Ca 2+ -dependent fluorescence changes, although with modest sensitivity compared to cytosolic controls. Second, we fused the genetically encoded calcium indicator GCaMP6m to the C terminus of BK (BK1082GCaMP). This construct preserved wild-type electrophysiological properties and displayed robust fluorescence responses to Ca 2+ elevations, with ΔF/F 0 values exceeding conventional membrane-tethered GCaMP. Patch-clamp fluorometry demonstrated simultaneous voltage- and Ca 2+ -dependent activity, while co-expression with NMDA receptors revealed faithful tracking of nanodomain Ca 2+ influx during receptor-channel coupling. Together, these results establish BK channels as versatile scaffolds for engineering nanodomain-targeted Ca 2+ sensors. While cpHalo insertions provide proof of principle, the GCaMP-fused construct offers strong potential as a functional and sensitive reporter of localized Ca 2+ dynamics. This approach opens new avenues for investigating the molecular logic of Ca 2+ signaling in neuronal circuits and its dysregulation in disease.
Yanes-Alonso et al. (Sun,) studied this question.