Genetically encoded calcium indicators (GECIs)

GECIs measure calcium, the key signal molecule underlying cell functions such as heart, vessel, and airway contraction, lung secretion, autonomic neurotransmission, and immunocyte function.  GCaMP, originally developed by Junichi Nakai [1] is an example of a GECI.  It is a fusion of circularly permutated green fluorescent protein (GFP), calmodulin (CaM) and M13, a peptide sequence from myosin light chain kinase.  At the N terminal end of GCaMP, the M13 domain of myosin is linked to the C terminal half of GFP which in turn is linked to the N terminal half of GFP followed at the C terminal end of the molecule by calmodulin.  Calmodulin is a symmetrical, hinge-like protein that binds to four calcium ions via E-F motifs.

In the absence of calcium, the circularly permutated GFP exists in a poorly fluorescent state due to a water pathway that enables protonation of the chromophore and poor absorbance at the excitation wavelengths.  When calcium is present, CaM undergoes a conformational change, and the hinge region is able to bind helical peptide chains on target proteins, including M13.  This conformational change brings the C terminal and N terminal ends of GFP back in close proximity to each other resulting in rapid de-protonation of the chromophore, and bright fluorescence.[2,3]

The original GCaMP molecule designed and created by Junichi Nakai has been modified and improved and we are now utilizing 3rd generation molecules with higher dynamic ranges and more rapid off kinetics.  In addition, red shifted variants (RCaMP) have been developed allowing for discrete spectra when compared with GCaMP and can be used effectively in dual imaging experiments.[4]   Lastly, ER-GCaMP1 has been developed, allowing for calcium measurements in the endoplasmic reticulum.



Crystal structure of GCaMP in calcium bound state and calcium free state:

  1. Nakai, J., Ohkura, M., and Imoto, K. A high signal-to-noise Ca(2+) probe composed of a single fluorescent protein. Nature Biotechnol. 19:137-141, 2001.
  2. Wang, Q. et. al. “Structural basis for calcium sensing by GCaMP2”. Structure 16:1817-1827, 2008
  3. Akerboom J., et. al. Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. J. Biol. Chem. 284:6455-6464, 2009.
  4. Zhao, Y. et. al. “An expanded palette of genetically encoded Ca²⁺ indicators”. Science 333:1888-1891, 2011.

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