Extending the Color Palette of In Vivo Calcium Sensing

The state-of-the-art calcium indicator used in neurobiology today is the GFP-based sensor GCaMP3. GCaMP3 consists of a circularly permuted GFP, with attached to the N- and C- terminus the M13 peptide and the caclium binding protein calmodulin respectively. In the presence of calcium, calcium bound calmodulin changes conformation dramatically resulting in a high affinity for the M13 peptide. The protein closes the circular permutation site close to the GFP chromophore by the calmodulin-M13 peptide binding, and fluorescence is restored. Although this genetically encoded calcium indicator (GECI) has shown to be indispensable for in vivo calcium imaging in several different neural circuits in many model organisms, drawbacks exist in having only a single color green available. These drawbacks are spectral overlap with the optogenetical protein channelrhodopsin, difficulties in labeling different cell types, difficulties in using GECIs in GFP-transgenic animals and problematic light scattering effects when using green light.

To resolve this problems we set out to generate a family of different colored calcium indicators, including blue, cyan, yellow and the most sought for calcium indicator, red. We used different approaches to generate the different colors; for yellow, cyan and blue we mutagenized the existing GCaMP3 protein, for the red protein termed RCaMP we used a different fluorescent protein in the scaffold as indicator.

To aid our design methods we solved several crystal structures of the generated RCaMP protein, as well as the fluorescent protein used in our scaffold design. The random- and directed mutagenesis strategies used to generate a functional red calcium indicator are discussed, and applications in different model organisms of these calcium indicators are presented.

These new calcium indicators will open up a whole new venue of possibilities for people working in in vivo calcium imaging.