Single fluorescent protein-based Ca2+ sensors with increased dynamic range

• Published in: BMC biotechnology 2007-06, Vol.7 (1), p.37-37, Article 37
• Main Authors: Souslova, Ekaterina A, Belousov, Vsevolod V, Lock, John G, Strömblad, Staffan, Kasparov, Sergey, Bolshakov, Alexey P, Pinelis, Vsevolod G, Labas, Yulii A, Lukyanov, Sergey, Mayr, Lorenz M, Chudakov, Dmitriy M
• Format: Article
• Language: English
• Subjects: Animals; Calcium - metabolism; Fluorescent Dyes - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HeLa Cells; Humans; Medicin och hälsovetenskap; Mutagenesis, Site-Directed; PC12 Cells; Rats; Recombinant Fusion Proteins - metabolism; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Fluorescence - methods
• ISSN: 1472-6750; 1472-6750
• DOI: 10.1186/1472-6750-7-37

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 Ca2+ 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). Here we report significant progress on the development of the latter type of Ca2+ 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 Ca2+ 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 Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. 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.