Single fluorescent protein-based Ca(2+ )sensors with increased dynamic range

BACKGROUND: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca(2+ )sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). RESULTS: Here we report significant progress on the development of the latter type of Ca(2+ )sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca(2+ )concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca(2+)-free and Ca(2+)-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca(2+ )response to a prolonged glutamate treatment in cortical neurons. CONCLUSION: We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.