Genetically-Encoded Calcium Indicators

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Background

[Here an introduction about GECIs, some historical background, rationale etc]

FRET-based GECIs

Legend here. From [1]

In these probes, a calcium binding-sensitive conformational change is used to bring into close proximity two spectral variants of GFP, allowing Förster resonance energy transfer (FRET) to occur between these proteins. FRET results in an increase in the emission ratio of the probe (emission of the acceptor/emission of the donor).

FRET GECIs Employing Calmodulin

Cameleons were the first GFP-based GECIs to be developed. In these probes, the Ca2+ sensitive binding of calmodulin (CaM) to the M13 peptide (a peptide isolated from the myosin light chain kinase) is used bring together the two GFP variants.

[OUTDATED PROBES SHOULD BE GRAYED]

Acronym/Name Dynamic Range* Affinity (Kd**) Kinetics Other Info Refs
FIP-CBsm [2]
Cameleon-2 [2]
YC2.60 [2]
YC3.60 [2]
YC4.60 [2]
YC6.1 []
YC-Nano140 [3]
YC-Nano65 [3]
YC-Nano50 [3]
YC-Nano30 [3]
YC-Nano15 [3]
D3cpVenus (D3cpv) 5 x ratio 600 nM [4]

* See glossary. Here the dynamic range is the maximal fold change in the FRET ratio (simply called ratio in the table) upon Ca2+ binding.

** Kd = dissociation constant. See glossary.

FRET GECIs Employing Troponin C

[OUTDATED PROBES SHOULD BE GRAYED]

Acronym/Name Dynamic Range* Affinity (Kd) Kinetics Other Info Refs
TN-L15
TN-humTnC
TN-XL
TN-XXL

Single Fluorescein Protein (Single FP) GECIs

Single FP GECIs Employing a circularly permutated fluorescent protein (cpFP)

On binding Ca2+, CaM executes a conformational change, interacting with a CaM binding peptide (here M13) and altering the protonation state of the chromophore, thus changing the fluorescence intensity of the protein. From [1]

Pericam

[OUTDATED PROBES SHOULD BE GRAYED]

Acronym/Name Dynamic Range* Affinity (Kd) Kinetics Other Info Refs
Pericam
Flash-Pericam
Ratiometric Pericam
Inverse Pericam

GCaMPs and GCaMP variants

GCaMP probes are composed of a single circularly permutated GFP sandwiched between the M13 peptide and CaM. Several versions of the original GCaMP sensor have been published. The Ca2+-bound and Ca2+-free structures of GCaMP2 have resolved, allowing structure-guided mutagenesis of the probe, leading to GCaMP3. Recent versions include GCaMP4.1, which was used to image Xenopus gastrulation, but no sequence information or comparison with other GECIs is published. GCaMP-HS consists of GCaMP2 with a subset of the "superfolder GFP" mutations and was used for imaging zebrafish motor neurons, but was also not compared with other sensors. The G-GECO sensors were created from GCaMP3 by random mutagenesis; they show 2ϫ greater fluorescence increase in purified protein (Ca2+-saturated vs Ca2+-free) but were not tested in neurons. Recently, GCaMP5 was published and tested in various different animal models.

[OUTDATED PROBES SHOULD BE GRAYED]

Acronym/Name Dynamic Range* Affinity (Kd) Kinetics** Peak Excitation Wavelength (nm) Peak Emission Wavelength (nm) Available from Refs
Cytosolic GCaMPs
GCaMP [5]
GCaMP2 3-5x (purified) 840 nM (purified) [6]
GCaMP3 12x (purified) 660 nM (purified) ~510 [7]
GCaMP5 Unpublished
G-GECO0.5 20x 957 nM [8]
G-GECO1 25x [8]
G-GECO1.1 26x [8]
G-GECO1.2 23x [8]
B-GECO1 7x [8]
R-GECO1 16x [8]
GEM-GECO1 110x [8]
GEX-GECO1 26x [8]
GCaMPs targeted to subcellular compartments
SyGCaMP2 Addgene [9]
Lck-GCaMP2 [10]

* See glossary. Here the dynamic range is the maximal fluorescence fold change upon Ca2+ binding.

Grafted Sensors

Grafted sensors utilizing EF-hands or portions of CaM inserted into a fluorescent protein. Binding of Ca2+ causes a change in protein conformation and a shift in the protonation state of the chromophore. From [1]

Camgaroos

[OUTDATED PROBES SHOULD BE GRAYED]

Acronym/Name Dynamic Range* Affinity (Kd) Kinetics Other Info Refs
Camgaroo
Camgaroo 2

Bioluminescent Proteins

Aequorin

Upon binding of Ca2+, the aequorin undergoes a conformational change, releasing coelenteramide and emitting blue light. From [1]

Glossary

  • Dynamic range: the range of the signal levels that can be reliably measured. Example: a protein sensing the compound X will only be able to reliably report changes in the concentration of X within a certain range of concentrations. More prosaically, the dynamic range of a fluorescent sensor is often expressed as the maximal fluorescence fold change upon binding to X in the case of single FP sensors and the maximal FRET ratio fold change in the case of FRET-based sensors.

Properties of available GECIs

List of available constructs

Name Description Map Lab Addgene
GCaMP3 Looger Lab Plasmid 22692
GCaMP5 Looger Lab Plasmid 31788

List of available transgenic lines

Strain Name Type Description Lab Jackson Labs stock # Ref
GCaMP2
STOCK Tg(Myh6*/tetO-GCaMP2)1Mik/J This line has the expression of GCaMP2 directed to heart cells by a minimally active mouse alpha myosin heavy chain promoter (Myh6) fused to seven copies of the tetracycline-responsive promoter element (tetO). Michael Kotlikoff 012477
GCaMP3
B6;129S-Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J These Ai38 mice conditionally express GCaMP3 from the endogenous Gt(ROSA)26Sor locus. Expression is enhanced by the presence of a CAG promoter. Following Cre-mediated removal of the STOP cassette, GCaMP3 expression (EGFP fluorescence) is detected in the cre-expressing tissues. Hongkui Zeng, Allen Institute for Brain Science 014538

Links

Links

  • The GECI Project (HHMI Janelia Farm Campus), a collaborative effort to engineer and distribute improved and diversified genetically-encoded calcium indicators.

References

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  1. Error fetching PMID 18848629: [McCombs2008]
  2. Error fetching PMID 15247428: [Nagai2004]
  3. Error fetching PMID 20693999: [Horikawa2010]
  4. Error fetching PMID 16720273: [Palmer2006]
  5. Error fetching PMID 11175727: [Nakai2001]
  6. Error fetching PMID 16537386: [Tallini2006]
  7. Error fetching PMID 19898485: [Tian2009]
  8. Error fetching PMID 19898484: [Dreosti2009]
  9. Error fetching PMID 20495558: [Shigetomi2010]
  10. Error fetching PMID 18447377: [Man2008]
  11. Error fetching PMID 19461949: [Tian2008]
All Medline abstracts: PubMed HubMed