genetically encoded voltage indicator

Even though the idea of optical measurement of neuronal activity was proposed in the late 1960s,{{Cite journal|vauthors=Cohen LB, Keynes RD, Hille B|date=1968|title=Light scattering and birefringence changes during nerve activity|journal=Nature|volume=218|issue=5140|pages=438–441|doi=10.1038/218438a0|pmid=5649693|bibcode=1968Natur.218..438C |s2cid=4288546}} 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,{{Cite journal|vauthors=Siegel MS, Isacoff EY|date=1997|title=A genetically encoded optical probe of membrane voltage|journal=Neuron|volume=19|issue=4|pages=735–741|doi=10.1016/S0896-6273(00)80955-1|pmid=9354320|doi-access=free}} was constructed by fusing a modified green fluorescent protein with a voltage-sensitive K⁺ channel (Shaker). Unlike fluorescent proteins, the discovery of new GEVIs are seldom inspired by 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 research using directed evolution have shown promising results in GEVI optimization.{{Cite journal|display-authors=3|vauthors=Platisa J, Vasan G, Yang A, Pieribone VA|date=2017|title=Directed Evolution of Key Residues in Fluorescent Protein Inverses the Polarity of Voltage Sensitivity in the Genetically Encoded Indicator ArcLight|journal=ACS Chem. Neurosci.|volume=8|issue=3|pages=513–523|doi=10.1021/acschemneuro.6b00234|pmc=5355904|pmid=28045247}}

Conceptually, a GEVI should sense the voltage difference across the cell membrane and report it by a change in fluorescence. Many different structures can be used for the voltage sensing function,{{Cite journal|vauthors=Gong Y|date=2015|title=The evolving capabilities of rhodopsin-based genetically encoded voltage indicators|journal=Curr. Opin. Chem. Biol.|volume=27|pages=84–89|doi=10.1016/j.cbpa.2015.05.006|pmc=4571180|pmid=26143170}} but one essential feature is that it must be imbedded in the cell membrane. Usually, the voltage-sensing domain (VSD) of a GEVI spans across the membrane, and is connected to the fluorescent protein (FP). However, it is not necessary that sensing and reporting must happen in different structures - see, for example, the Archons.

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 opsin 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.{{Cite journal|display-authors=3|vauthors=Murata Y, Iwasaki H, Sasaki M, Inaba K, Okamura Y|date=2005|title=Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor|journal=Nature|volume=435|issue=7046|pages=1239–1243|doi=10.1038/nature03650|pmid=15902207|bibcode=2005Natur.435.1239M |s2cid=4427755}} Some GEVIs may have similar components, but in different positions. 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.

1. {{note label|name|A|↑|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.{{Cite journal|vauthors=Yang HH, St-Pierre F|date=2016|title=Genetically Encoded Voltage Indicators: Opportunities and Challenges|journal=J. Neurosci.|volume=36|issue=39|pages=9977–9989|doi=10.1523/JNEUROSCI.1095-16.2016|pmc=5039263|pmid=27683896}} For now, no existing GEVI meets all the desired properties, so searching for a perfect GEVI is still a quite competitive research area.

Different types of GEVIs are being developed 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 has subcellular spatial resolution{{Cite journal|vauthors=Kaschula R, Salecker I|date=2016|title=Neuronal Computations Made Visible with Subcellular Resolution|journal=Cell|volume=166|issue=1|pages=18–20|doi=10.1016/j.cell.2016.06.022|pmid=27368098|doi-access=free}} and temporal resolution as low as 0.2 milliseconds, about an order of magnitude faster than calcium imaging. This allows for spike detection fidelity comparable to electrode-based electrophysiology but without the invasiveness. Researchers have used it to probe neural communications of an intact brain (of Drosophila{{Cite journal|display-authors=3|vauthors=Cao G, Platisa J, Pieribone VA, Raccuglia D, Kunst M, Nitabach MN|date=2013|title=Genetically targeted optical electrophysiology in intact neural circuits|journal=Cell|volume=154|issue=4|pages=904–913|doi=10.1016/j.cell.2013.07.027|pmc=3874294|pmid=23932121}} or mouse{{Cite journal|vauthors=Knöpfel T, Gallero-Salas Y, Song C|date=2015|title=Genetically encoded voltage indicators for large scale cortical imaging come of age|journal=Curr. Opin. Chem. Biol.|volume=27|pages=75–83|doi=10.1016/j.cbpa.2015.06.006|pmid=26115448}}), electrical spiking of bacteria (E. coli), and human stem-cell derived cardiomyocyte.{{Cite journal|display-authors=3|vauthors=Kaestner L, Tian Q, Kaiser E, Xian W, Müller A, Oberhofer M, Ruppenthal S, Sinnecker D, Tsutsui H, Miyawaki A, Moretti A, Lipp P|date=2015|title=Genetically Encoded Voltage Indicators in Circulation Research|journal=Int. J. Mol. Sci.|volume=16|issue=9|pages=21626–21642|doi=10.3390/ijms160921626|pmc=4613271|pmid=26370981|doi-access=free}}{{Cite journal|last1=Zhang|first1=Joe Z.|last2=Termglinchan|first2=Vittavat|last3=Shao|first3=Ning-Yi|last4=Itzhaki|first4=Ilanit|last5=Liu|first5=Chun|last6=Ma|first6=Ning|last7=Tian|first7=Lei|last8=Wang|first8=Vicky Y.|last9=Chang|first9=Alex C. Y.|last10=Guo|first10=Hongchao|last11=Kitani|first11=Tomoya|date=2019-05-02|title=A Human iPSC Double-Reporter System Enables Purification of Cardiac Lineage Subpopulations with Distinct Function and Drug Response Profiles|journal=Cell Stem Cell|language=en|volume=24|issue=5|pages=802–811.e5|doi=10.1016/j.stem.2019.02.015|issn=1934-5909|pmid=30880024|pmc=6499654|doi-access=free}}

Conversely, any form of voltage indication has inherent limitations.{{cite journal |title=Voltage imaging: pitfalls and potential

|last1=Kulkarni |first1=Rishikesh U |last2=Miller |first2=Evan W

|journal=Biochemistry

|volume=56

|number=39

|pages=5171--5177

|year=2017

|url=https://pubs.acs.org/doi/pdf/10.1021/acs.biochem.7b00490

|publisher=ACS Publications}} Imaging must be fast, or short voltage excursions will be missed. This means fewer photons per image. Next, the brightness is inherently less, as about a thousand-fold fewer voltage indicators can fit in the membrane, when compared a cytosolic sensor such as used in calcium imaging. Finally, since the sensor is bound to the membrane (as opposed to the cytosol), it can be ambiguous which cell is responding.

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Category:Neuroscience

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Category:Protein engineering

Category:Electrophysiology