GCaMP
From Wikipedia, the free encyclopedia
GCaMP is a genetically encoded calcium indicator (GECI) initially developed in 2001 by Junichi Nakai.[1] It is a synthetic fusion of green fluorescent protein (GFP), calmodulin (CaM), and M13, a peptide sequence from myosin light-chain kinase.[2] When bound to Ca2+, GCaMP fluoresces green with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm.[3] It is used in biological research to measure intracellular Ca2+ levels both in vitro and in vivo using virally transfected or transgenic cell and animal lines.[2][4] The genetic sequence encoding GCaMP can be inserted under the control of promoters exclusive to certain cell types, allowing for cell-type specific expression of GCaMP.[5] Since Ca2+ is a second messenger that contributes to many cellular mechanisms and signaling pathways, GCaMP allows researchers to quantify the activity of Ca2+-based mechanisms and study the role of Ca2+ ions in biological processes of interest.
GCaMP consists of three key domains: an M13 domain at the N-terminus, a calmodulin (CaM) domain at the C-terminus, and a GFP domain in the center. The GFP domain is circularly permuted such that the native N- and C-termini are fused together by a six-amino-acid linking sequence, and the GFP sequence is split in the middle, creating new N- and C-termini that connect to the M13 and CaM domains.[6]
In the absence of Ca2+, the GFP chromophore is exposed to water and exists in a protonated state with minimal fluorescence intensity. Upon Ca2+ binding, the CaM domain undergoes a conformational change and tightly binds to the M13 domain alpha helix, preventing water molecules from accessing the chromophore. As a result, the chromophore rapidly deprotonates and converts into an anionic form that fluoresces brightly, similar to native GFP.[7]
History and development
In 2001, Nakai et al. reported the development of GCaMP1 as a Ca2+ probe with improved signal-to-noise ratio compared to previously developed fluorescent Ca2+ probes.[1] The first transgenic mouse expressing GCaMP1 was reported in 2004.[5] However, at 37 ˚C (physiological temperature in mammals), GCaMP1 did not fold stably or fluoresce, limiting its potential use as a calcium indicator in vivo.[1][8]
In 2006, Tallini et al. subsequently reported the improvement of GCaMP1 to GCaMP2, which exhibited brighter fluorescence than GCaMP1 and greater stability at mammalian body temperatures. Tallini et al. expressed GCaMP2 in cardiomyocytes in mouse embryos to perform the first in vivo GCaMP imaging of Ca2+ in mammals.[8]
Further modifications of GCaMP, including GCaMP3, GCaMP5, GCaMP6, and jGCaMP7, have been developed to progressively improve the signal, sensitivity, and dynamic range of Ca2+ detection,[2][9][10][11] with recent versions exhibiting fluorescence similar to native GFP.[11]
Variants in use
Both slow variants (GCaMP6s, jGCaMP7s) and fast variants (GCaMP6f, jGCaMP7f) are used in biological and neuroscience research. The slow variants are brighter and more sensitive to small changes in Ca2+ levels, such as single action potentials; on the other hand, the fast variants are less sensitive but respond more quickly, making them useful for tracking changes in Ca2+ levels over precise timescales.[12][13] GCaMP6 also has a medium variant, GCaMP6m, whose kinetics are intermediate between GCaMP6s and GCaMP6f.[12] Other variants of jGCaMP7 are also employed: jGCaMP7b exhibits bright baseline fluorescence and is used for imaging dendrites and axons, while jGCaMP7c exhibits greater contrast between maximal and baseline fluorescence and is advantageous for imaging large populations of neurons.[12]
In 2018, Yang et al. reported the development of GCaMP-X, generated by the addition of a calmodulin-binding motif. Since the GCaMP calmodulin domain, when unbound, disrupts L-type calcium channel gating, the added calmodulin-binding motif prevents GCaMP-X from interfering with calcium-dependent signaling mechanisms.[14]
In 2020, Zhang et al. reported the development of jGCaMP8, including sensitive, medium, and fast variants, which exhibit faster kinetics and greater sensitivity than the corresponding jGCaMP7 variants.[15]
Red fluorescent indicators have also been developed: jRCaMP1a and jRCaMP1b use a circular permutation of the red fluorescent protein mRuby instead of GFP, while jRGECO1a is based on the red fluorescent protein mApple.[12][16] Since the blue light used to excite GCaMP is scattered by tissue and the emitted green light is absorbed by blood, red fluorescent indicators provide more penetration and imaging depth in vivo than GCaMP. Use of red fluorescent indicators also avoids the photodamage caused by blue excitation light.[16] Moreover, red fluorescent indicators allow for concurrent use of optogenetics, which is difficult with GCaMP because the excitation wavelengths of GCaMP overlap with those of channelrhodopsin-2 (ChR2).[16][17][18] Simultaneous use of red and green GECIs can provide two-color visualization of different subcellular regions or cell populations.[16][17][19]