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Genetically encoded voltage indicator

Genetically encoded voltage indicator (or GEVI) is a protein that can sense membrane potential in a cell and relate the change in voltage to a form of output, often fluorescent level.[1] It is a promising optogenetic recording tool that enables exporting electrophysiological signals from cultured cells, live animals, and ultimately human brain. Examples of notable GEVIs include ArcLight, ASAP1,[2] ASAP2s,[3] and Ace2N-mNeon.



Despite that the idea of optical measurement of neuronal activity was proposed in the late 1960s,[4] the first successful GEVI that was convenient enough to put into actual use was not developed until technologies of genetic engineering had become mature in the late 1990s. The first GEVI, coined FlaSh,[5] was constructed by fusing a modified green fluorescent protein with a voltage-sensitive K+ channel (Shaker). Unlike fluorescent proteins, the discovery of new GEVIs were seldomly inspired by the nature, for it is hard to find an organism which naturally has the ability to change its fluorescence based on voltage. Therefore, new GEVIs are mostly the products of genetic and protein engineering.

Two methods can be utilized to find novel GEVIs: rational design and directed evolution. The former method contributes to the most of new GEVI variants, but recent researches using directed evolution have shown promising results in GEVI optimization.[6]


GEVI can have many configuration designs in order to realize voltage sensing function.[7] An essential feature of GEVI structure is that it must situate on the cell membrane. Conceptually, the structure of a GEVI should permit the function of sensing the voltage difference and reporting it by change in fluorescence. Usually, the voltage-sensing domain (VSD) of a GEVI spans across the membrane, and is connected to the fluorescent protein(s). However, it is not necessary that sensing and reporting should happen in different structures, e.g. Arch.

By structure, GEVIs can be classified into four categories based on the current findings: (1) GEVIs contain a fluorescent protein FRET pair, e.g. VSFP1, (2) Single opsion GEVIs, e.g. Arch, (3) Opsin-FP FRET pair GEVIs, e.g. MacQ-mCitrine, (4) single FP with special types of voltage sensing domains, e.g. ASAP1. A majority of GEVIs are based on the Ciona intestinalis voltage sensitive phosphatase (Ci-VSP or Ci-VSD (domain)), which was discovered in 2005 from the genomic survey of the organism.[8] Some GEVIs might have similar components, but with different positioning of them. For example, ASAP1 and ArcLight both use a VSD and one FP, but the FP of ASAP1 is on the outside of the cell whereas that of ArcLight is on the inside, and the two FPs of VSFP-Butterfly are separated by the VSD, while the two FPs of Mermaid are relatively close to each other.

Table of GEVIs and their structure
GEVI[A] Year Sensing Reporting Precursor
FlaSh[5] 1997 Shaker (K+ channel) GFP -
VSFP1[9] 2001 Rat Kv2.1 (K+ channel) FRET pair: CFP and YFP -
SPARC[10] 2002 Rat Na+ channel GFP -
VSFP2's[11] 2007 Ci-VSD FRET pair: CFP (Cerulean) and YFP (Citrine) VSFP1
Flare[12] 2007 Kv1.4 (K+ channel) YFP FlaSh
VSFP3.1[13] 2008 Ci-VSD CFP VSFP2's
Mermaid[14] 2008 Ci-VSD FRET pair: Marine GFP (mUKG) and OFP (mKOκ) VSFP2's
hVOS[15] 2008 Dipicrylamine GFP -
Red-shifted VSFP's[16] 2009 Ci-VSD RFP/YFP (Citrine, mOrange2, TagRFP, or mKate2) VSFP3.1
PROPS[17] 2011 Modified green-absorbing proteorhodopsin (GPR) Same as left -
Zahra, Zahra 2[18] 2012 Nv-VSD, Dr-VSD FRET pair: CFP (Cerulean) and YFP (Citrine) VSFP2's
ArcLight[19] 2012 Ci-VSD Modified super ecliptic pHluorin -
Arch[20] 2012 Archaerhodopsin 3 Same as left -
ElectricPk[21] 2012 Ci-VSD Circularly permuted EGFP VSFP3.1
VSFP-Butterfly[22] 2012 Ci-VSD FRET pair: YFP (mCitrine) and RFP (mKate2) VSFP2's
VSFP-CR[23] 2013 Ci-VSD FRET pair: GFP (Clover) and RFP(mRuby2) VSFP2.3
Mermaid2[24] 2013 Ci-VSD FRET pair: CFP (seCFP2) and YFP Mermaid
Mac GEVIs[25] 2014 Mac rhodopsin (FRET acceptor) FRET doner: mCitrine, or mOrange2 -
QuasAr1, QuasAr2[26] 2014 Modified Archaerhodopsin 3 Same as left Arch
Archer[27] 2014 Modified Archaerhodopsin 3 Same as left Arch
ASAP1[2] 2014 Modified Gg-VSD Circularly permuted GFP -
Ace GEVIs[28] 2015 Modified Ace rhodopsin FRET doner: mNeonGreen Mac GEVIs
ArcLightning[29] 2015 Ci-VSD Modified super ecliptic pHluorin ArcLight
Pado[30] 2016 Voltage-gated proton channel Super ecliptic pHluorin -
ASAP2f[31] 2016 Modified Gg-VSD Circularly permuted GFP ASAP1
FlicR1[32] 2016 Ci-VSD Circularly permuted RFP (mApple) VSFP3.1
Bongwoori[33] 2017 Ci-VSD Modified super ecliptic pHluorin ArcLight
ASAP2s[3] 2017 Modified Gg-VSD Circularly permuted GFP ASAP1
ASAP-Y[34] 2017 Modified Gg-VSD Circularly permuted GFP ASAP1
(pa)QuasAr3(-s)[35][36][37] 2019 Modified Archaerhodopsin 3 Same as left QuasAr2
  1. Names in italic denote GEVIs not named.


A GEVI can be evaluated by its many characteristics. These traits can be classified into two categories: performance and compatibility. The performance properties include brightness, photostability, sensitivity, kinetics (speed), linearity of response, etc., while the compatibility properties cover toxicity (phototoxicity), plasma membrane localization, adaptability of deep-tissue imaging, etc.[38] For now, no existing GEVI meets all the desired properties, so searching for a perfect GEVI is still a quite competitive research area.

Applications and advantagesEdit

Different types of GEVIs are seen being used in many biological or physiological research areas. It is thought to be superior to conventional voltage detecting methods like electrode-based electrophysiological recordings, calcium imaging, or voltage sensitive dyes. It can show neuron signals with subcellular spatial resolution.[39] It has fast temporal resolution (sub-millisecond[28]), matching or surpassing that of the electrode recordings, and about one magnitude faster than calcium imaging. Researchers have used it to probe neural communications of an intact brain (of Drosophila[40] or mouse[41]), electrical spiking of bacteria (E. coli[17]), and human stem-cell derived cardiomyocyte.[42]


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