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

Abstract

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 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).

Results: 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.

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.

Figure 1

Proposed model of a spatial organization of Pericams and GcaMPs. a. Sensor constructs scheme. b. Proposed positional relationship of key amino acid residues (145–148) and sensitive domains (calmodulin and M13) within Ca²⁺ sensors.

Figure 2

Spectral characteristics of Case12 (a,c,e,g) and Case16 (b,d,f,h). a,b. Maximum fluorescent responses. Fluorescent spectra recorded in the presence of 0.5 mM EGTA (dashed lines) or 1 mM Ca²⁺ (solid lines) at pH 7.4. c,d. Absorbance in the presence of 0.5 mM EGTA (dashed lines) or 0.4 mM Ca²⁺ (solid lines) at pH 7.4. e,f. Dependence of sensors fluorescence on pH in the presence of 0.5 mM EGTA (dashed lines) or of 0.2 mM Ca²⁺ (solid lines). g,h. Ca²⁺ titration curves, at pH 7.4.

Figure 3

Case12 and Case16 in living cells. a,b. Typical responses of HeLa cells expressing Case12 (a) and Case16 (b) to Ca²⁺ ionophore A23187. c,d. HeLa cells expressing Case12 are shown before (b) and after (c) ionophore addition. e-h. Fluorescence changes of M21 (human Melanoma-derived) cells expressing Case12 in response to 100 μM ATP. Images were captured every 0.294 sec on the confocal microscope. e,f. Individual responses of two selected cells within 400 s after ATP addition. g,h. The same cells, first 60 s of response. i. PC12 cells response to 500 uM carbachol (CCH). j. PC12 cells response to 30 mM KCl. For i and j first and second arrows indicate the moments of a compound addition and of washing start, respectively.

Figure 4

Ca²⁺ response to a prolonged glutamate treatment in cortical neurons. Fluorescent signals of Case12 (gray solid line, excitation at 490 nm, 515–565 nm emission filter) and fura-2FF (black dashed line, ratio of 340 nm and 380 nm excited green fluorescence, 505–530 nm emission filter) are shown.