A Genetically Encoded Calcium Ion Biosensor with an Exceptionally Large Ratiometric Response

The objective of this work was to overcome some of the long-standing limitations of ratiometric fluorescent protein (FP)-based Ca2+ biosensors, which typically rely on Förster resonance energy transfer (FRET) between two FPs and generally exhibit only relatively modest Ca2+-dependent changes in emission ratio. To develop biosensors with substantially greater ratiometric changes, we explored an alternative biosensor design strategy in which two independently optimized intensiometric single FP-based Ca2+ biosensors were hybridized into a single protein construct such that they employed a shared calmodulin (CaM) and CaM-binding peptide (CBP). By hybridizing a direct-response red FP-based biosensor with an inverse-response green FP-based biosensor, we created SuiCa, a single-polypeptide Ca2+ biosensor that exhibits exceptionally large red-to-green ratiometric fluorescence changes as purified protein (∼60-fold) and when expressed in immortalized cell cultures (∼80-fold) and primary neurons (∼37-fold). Relative to co-expression of two spectrally distinct FP-based Ca2+ biosensors, SuiCa provides the advantages of a smaller gene size, a fixed fluorophore stoichiometry, and a ratiometric response that depends on Ca2+ binding to a single, shared, CaM plus CBP domain. With these advantages, along with its bright fluorescence and large ratiometric change, SuiCa represents a new addition to the Ca2+ imaging toolbox.