Single Fluorescent Protein-Based Ca2+ Sensors With Increased Dynamic Range

BMC Biotechnol. 2007 Jun 29;7:37. doi: 10.1186/1472-6750-7-37.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Fluorescent Dyes / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Mutagenesis, Site-Directed
  • PC12 Cells
  • Rats
  • Recombinant Fusion Proteins / metabolism*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / methods*

Substances

  • Fluorescent Dyes
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Calcium