Genetically encoded voltage indicator (or GEVI) is a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
that can sense
membrane potential
Membrane potential (also transmembrane potential or membrane voltage) is the difference in electric potential between the interior and the exterior of a biological cell. It equals the interior potential minus the exterior potential. This is th ...
in a cell and relate the change in
voltage
Voltage, also known as (electrical) potential difference, electric pressure, or electric tension, is the difference in electric potential between two points. In a Electrostatics, static electric field, it corresponds to the Work (electrical), ...
to a form of output, often
fluorescent level. It is a promising
optogenetic recording tool that enables recording of
signals from cultured cells and live animals. Examples of GEVI families include Quasar/Archon, Ace-mNeon, and ASAP.
History
Even though 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
The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victo ...
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
In chemical biology and biomolecular engineering, rational design (RD) is an umbrella term which invites the strategy of creating new molecules with a certain functionality, based upon the ability to predict how the molecule's structure (specific ...
and
directed evolution
Directed evolution (DE) is a method used in protein engineering that mimics the process of natural selection to steer proteins or nucleic acids toward a user-defined goal. It consists of subjecting a gene to iterative rounds of mutagenesis (cre ...
. The former method contributes to the most of new GEVI variants, but recent research using directed evolution have shown promising results in GEVI optimization.
Structure
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, 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
''Ciona intestinalis'' (sometimes known by the common name of vase tunicate) is an ascidian (sea squirt), a tunicate with very soft tunic. Its Latin name literally means "pillar of intestines", referring to the fact that its body is a soft, tran ...
''
voltage sensitive phosphatase
Voltage sensitive phosphatases or voltage sensor-containing phosphatases, commonly abbreviated VSPs, are a protein family found in many species, including humans, mice, zebrafish, frogs, and sea squirt.
Discovery
The first voltage sensitive phos ...
(Ci-VSP or Ci-VSD (domain)), which was discovered in 2005 from the
genomic
Genomics is an interdisciplinary field of molecular biology focusing on the structure, function, evolution, mapping, and editing of genomes. A genome is an organism's complete set of DNA, including all of its genes as well as its hierarchical, ...
survey of the organism. 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.
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 (speed), linearity of response, etc., while the compatibility properties cover toxicity (
phototoxicity
Phototoxicity, also called photoirritation, is a chemically induced skin irritation, requiring light, that does not involve the immune system. It is a type of photosensitivity.
The skin response resembles an exaggerated sunburn. The involved chem ...
), plasma membrane localization, adaptability of deep-tissue imaging, etc.
Applications, advantages, and disadvantages
Different types of GEVIs are being developed in many biological or physiological research areas. Unlike earlier voltage detecting methods like
electrode-based electrophysiological recordings or
voltage sensitive dyes, GEVIs can be expressed stably, and can be targeted to particular cell types. GEVIs have subcellular spatial resolution and temporal resolution as low as 0.2 milliseconds, at least 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 them to probe neural communications of an intact brain (of ''
Drosophila
''Drosophila'' (), from Ancient Greek δρόσος (''drósos''), meaning "dew", and φίλος (''phílos''), meaning "loving", is a genus of fly, belonging to the family Drosophilidae, whose members are often called "small fruit flies" or p ...
'' or mouse), electrical spiking of
bacteria
Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...
(''
E. coli
''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...
''
), and human stem-cell derived
cardiomyocyte
Cardiac muscle (also called heart muscle or myocardium) is one of three types of vertebrate muscle tissues, the others being skeletal muscle and smooth muscle. It is an involuntary, striated muscle that constitutes the main tissue of the wall of ...
.
Conversely, any form of voltage indication has inherent limitations.
Imaging must be fast, or short voltage excursions will be missed. This means fewer photons per image exposure. Next, brightness per cell is inherently lower than calcium indicators, as about a 30-fold fewer voltage indicators can fit in the membrane compared to cytosolic calcium indicators.
References
{{Optogenetics
Neuroscience
Biotechnology
Protein engineering
Electrophysiology