Genetically encoded voltage indicator


Genetically encoded voltage indicator 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. 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, ASAP3, and Ace2N-mNeon.

History

Despite that the idea of optical measurement of neuronal activity was proposed in the late 1960s, 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, was constructed by fusing a modified green fluorescent protein with a voltage-sensitive K+ channel. 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.

Structure

GEVI can have many configuration designs in order to realize voltage sensing function. 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 of a GEVI spans across the membrane, and is connected to the fluorescent protein. 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: GEVIs contain a fluorescent protein FRET pair, e.g. VSFP1, Single opsin GEVIs, e.g. Arch, Opsin-FP FRET pair GEVIs, e.g. MacQ-mCitrine, 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, which was discovered in 2005 from the genomic survey of the organism. 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.

GEVIYearSensingReportingPrecursor
FlaSh1997Shaker GFP-
VSFP12001Rat Kv2.1 FRET pair: CFP and YFP-
SPARC2002Rat Na+ channelGFP-
VSFP2's2007Ci-VSDFRET pair: CFP and YFP VSFP1
Flare2007Kv1.4 YFPFlaSh
VSFP3.12008Ci-VSDCFPVSFP2's
Mermaid2008Ci-VSDFRET pair: Marine GFP and OFP VSFP2's
hVOS2008DipicrylamineGFP-
Red-shifted VSFP's2009Ci-VSDRFP/YFP VSFP3.1
PROPS2011Modified green-absorbing proteorhodopsin Same as left-
Zahra, Zahra 22012Nv-VSD, Dr-VSDFRET pair: CFP and YFP VSFP2's
ArcLight2012Ci-VSDModified super ecliptic pHluorin-
Arch2012Archaerhodopsin 3Same as left-
ElectricPk2012Ci-VSDCircularly permuted EGFPVSFP3.1
VSFP-Butterfly2012Ci-VSDFRET pair: YFP and RFP VSFP2's
VSFP-CR2013Ci-VSDFRET pair: GFP and RFPVSFP2.3
Mermaid22013Ci-VSDFRET pair: CFP and YFPMermaid
Mac GEVIs2014Mac rhodopsin FRET doner: mCitrine, or mOrange2-
QuasAr1, QuasAr22014Modified Archaerhodopsin 3Same as leftArch
Archer2014Modified Archaerhodopsin 3Same as leftArch
ASAP12014Modified Gg-VSDCircularly permuted GFP-
Ace GEVIs2015Modified Ace rhodopsinFRET doner: mNeonGreenMac GEVIs
ArcLightning2015Ci-VSDModified super ecliptic pHluorinArcLight
Pado2016Voltage-gated proton channelSuper ecliptic pHluorin-
ASAP2f2016Modified Gg-VSDCircularly permuted GFPASAP1
FlicR12016Ci-VSDCircularly permuted RFP VSFP3.1
Bongwoori2017Ci-VSDModified super ecliptic pHluorinArcLight
ASAP2s2017Modified Gg-VSDCircularly permuted GFPASAP1
ASAP-Y2017Modified Gg-VSDCircularly permuted GFPASAP1
QuasAr32019Modified Archaerhodopsin 3Same as leftQuasAr2
Voltron2019Modified Ace rhodopsin FRET doner: Janelia Fluor -
ASAP32019Modified Gg-VSDCircularly permuted GFPASAP2s


Characteristics

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, linearity of response, etc., while the compatibility properties cover toxicity, plasma membrane localization, adaptability of deep-tissue imaging, etc. 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 advantages

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. It has fast temporal resolution, 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, electrical spiking of bacteria, and human stem-cell derived cardiomyocyte.