Channelrhodopsins are a subfamily of
opsinOpsins are a group of light-sensitive 35–55 kDa membrane-bound G protein-coupled receptors of the retinylidene protein family found in photoreceptor cells of the retina. Five classical groups of opsins are involved in vision, mediating the conversion of a photon of light into an electrochemical...
proteinProteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s that function as
light-gated ion channelLight-gated ion channels are a group of transmembrane proteins that form ion channels; pores which open or close in response to light. Most light-gated ion channels have been synthesized in the laboratory for study, though one naturally occurring example, Channelrhodopsin, is currently known...
s. They serve as
sensorySensory may refer to:relating to senses or smellIn biology:* Sensory preference* Sensory system, part of the nervous system of organisms* Sensory neuron, nerve cell responsible for transmitting information about external stimuli...
photoreceptorThe eyespot apparatus is a photoreceptive organelle found in the flagellate cells of green algae and other unicellular photosynthetic organisms such as euglenids. It allows the cells to sense light direction and intensity and respond to it by swimming either towards the light or away from the...
s in unicellular
green algaeThe green algae are the large group of algae from which the embryophytes emerged. As such, they form a paraphyletic group, although the group including both green algae and embryophytes is monophyletic...
, controlling
phototaxisPhototaxis is a kind of taxis, or locomotory movement, that occurs when a whole organism moves in response to the stimulus of light. This is advantageous for phototrophic organisms as they can orient themselves most efficiently to receive light for photosynthesis...
, i.e. movement in response to light. Expressed in cells of other organisms, they enable the use of light to control
intracellularNot to be confused with intercellular, meaning "between cells".In cell biology, molecular biology and related fields, the word intracellular means "inside the cell".It is used in contrast to extracellular...
acidity,
calciumCalcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
influx,
electrical excitabilityMembrane potential is the difference in electrical potential between the interior and exterior of a biological cell. All animal cells are surrounded by a plasma membrane composed of a lipid bilayer with a variety of types of proteins embedded in it...
, and other cellular processes. Three channelrhodopsins are currently known: Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2), and Volvox Channelrhodopsin (VChR1). All known Channelrhodopsins are nonspecific cation channels, conducting H
+, Na
+, K
+, and Ca
2+ ions.
History
Channelrhodopsins were first discovered to be a single-component light-activated cation channel from the green
algaeAlgae are a large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms, such as the giant kelps that grow to 65 meters in length. They are photosynthetic like plants, and "simple" because their tissues are not organized into the many...
Chlamydomonas reinhardtiiChlamydomonas reinhardtii is a single celled green alga about 10 micrometres in diameter that swims with two flagella. They have a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and an "eyespot" that senses light.Although widely distributed...
by Georg Nagel and Peter Hegemann in 2002. Channelrhodopsin-2 (ChR2), which was also isolated from
Chlamydomonas reinhardtiiChlamydomonas reinhardtii is a single celled green alga about 10 micrometres in diameter that swims with two flagella. They have a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and an "eyespot" that senses light.Although widely distributed...
, was similarly discovered by Peter Hegemann's group in 2001, and submitted directly to
GenBankThe GenBank sequence database is an open access, annotated collection of all publicly available nucleotide sequences and their protein translations. This database is produced and maintained by the National Center for Biotechnology Information as part of the International Nucleotide Sequence...
, before being characterized by Georg Nagel's group and discovered independently by John Spudich's group in 2002. In collaboration with Peter Hegemann, along with Ernst Bamberg and with Georg Nagel as first and corresponding author, it was demonstrated that channelrhodopsin-2 is also a directly light-gated ion channel like ChR1.
In 2005, three groups sequentially established ChR2 as a tool for
geneticallyGenetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
targeted optical remote control (
optogeneticsOptogenetics is the combination of genetic and optical methods to control specific events in targeted cells of living tissue, even within freely moving mammals and other animals, with the temporal precision needed to keep pace with functioning intact biological systems.In 2010, optogenetics was...
) of
neuronA neuron is an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Chemical signaling occurs via synapses, specialized connections with other cells. Neurons connect to each other to form networks. Neurons are the core components of the nervous...
s, neural circuits and behavior. At first, Karl Deisseroth's lab (in a paper published in August 2005) demonstrated that ChR2 could be deployed to control
mammalMammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian neurons
in vitroIn vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
, achieving temporal precision on the order of milliseconds (both, in terms of delay to spiking, as well as in terms of temporal jitter). This was a significant finding, since firstly, all opsins (microbial as well as vertebrate) require
retinalRetinal, also called retinaldehyde or vitamin A aldehyde, is one of the many forms of vitamin A . Retinal is a polyene chromophore, and bound to proteins called opsins, is the chemical basis of animal vision...
as the light-sensing co-factor, and it was unclear, if central mammalian nerve cells would contain sufficient retinal levels - but surprisingly, they do. Secondly, it showed despite the small single-channel conductance, sufficient potency to drive mammalian neurons above action potential threshold. Thirdly, it demonstrated channelrhodopsin to be the first optogenetic tool, with which neural activity could be controlled with the temporal precision at which neurons operate (a few milliseconds); an earlier tool for photostimulation, cHARGe, demonstrated proof of principle in cultured neurons but was never usable since it operated with a precision on the order of seconds, was highly variable, and did not allow control of individual
action potentialIn physiology, an action potential is a short-lasting event in which the electrical membrane potential of a cell rapidly rises and falls, following a consistent trajectory. Action potentials occur in several types of animal cells, called excitable cells, which include neurons, muscle cells, and...
s.
Five months later (December 2005), a second study was published by Peter Hegemann's and Stefan Herlitze's groups confirming the ability of ChR2 to control the activity of
vertebrateVertebrates are animals that are members of the subphylum Vertebrata . Vertebrates are the largest group of chordates, with currently about 58,000 species described. Vertebrates include the jawless fishes, bony fishes, sharks and rays, amphibians, reptiles, mammals, and birds...
neurons, at this time in the chick spinal cord. This study was also the first, where ChR2 was expressed alongside an optical silencer, vertebrate
rhodopsinRhodopsin, also known as visual purple, is a biological pigment of the retina that is responsible for both the formation of the photoreceptor cells and the first events in the perception of light. Rhodopsins belong to the G-protein coupled receptor family and are extremely sensitive to light,...
-4 in this case, demonstrating for the first time, that excitable cells could be activated and silenced using these two tools simultaneously, but illuminating the tissue at different wavelengths. In the same month, the groups of Alexander Gottschalk and Ernst Bamberg (with Georg Nagel taking the experimental lead) demonstrated that ChR2, if expressed in specific neurons or muscle cells, can evoke predictable behaviours, i.e. can control the nervous system of an intact animal, which in this case was the invertebrate
C. elegansCaenorhabditis elegans is a free-living, transparent nematode , about 1 mm in length, which lives in temperate soil environments. Research into the molecular and developmental biology of C. elegans was begun in 1974 by Sydney Brenner and it has since been used extensively as a model...
. This was the first study, using the remote control via ChR2 to steer the behaviour of an animal. And it was also the first study, that deployed ChR2 for an optogenetic experiment - i.e. that rendered genetically specified cell types subject to optical remote control, while the other two studies, did not specifically target ChR2 expression genetically. Although both aspects had been illustrated earlier that year by another group, the
MiesenböckGero Miesenböck is Waynflete Professor of Physiology at the University of Oxford and a fellow of Magdalen College. A native of Austria, he received his M.D. from the University of Innsbruck and undertook postdoctoral training with James Rothman...
lab, deploying the indirectly light-gated ion channel P2X2, it was henceforth microbial opsins like channelrhodopsin that dominated the field of genetically targeted remote control of excitable cells, due to the power, speed, targetability, ease of use, and temporal precision of direct optical activation, not requiring any external chemical compound such as caged ligands.
To overcome its principle downsides - the small single-channel conductance (especially in steady-state), the fixation on one optimal excitation wavelength (~470 nm, blue) as well as the relatively long recovering time, not permitting controlled firing of neurons above 20–40 Hz - ChR2 has been optimized using
genetic engineeringGenetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
: The first optimization involved the
point mutationA point mutation, or single base substitution, is a type of mutation that causes the replacement of a single base nucleotide with another nucleotide of the genetic material, DNA or RNA. Often the term point mutation also includes insertions or deletions of a single base pair...
H134R (exchanging the amino acid Histidine in position 134 of the native protein for an Arginine), and published in the same paper, that established ChR2 as an optogenetic tool in
C. elegans. Further improvements were mainly carried by the close collaboration between the groups of Peter Hegemann and Karl Deisseroth. They included the introduction of the point mutation C128S, which made the resulting ChR2-derivative a step-function tool, that stays in the open state once "switched on" by blue light, until it is switched off by yellow light - a modification that deteriorates temporal precision, but greatly increases light sensitivity (by two orders of magnitude). One year later (2010) the same authors introduced another mutation into the native molecule, E123T, yielding the ChR2-derivative ChETA, which does not only feature a higher single-channel conductance (in the desensitized/steady state), but also a much faster on- and off-
kineticsChemical kinetics, also known as reaction kinetics, is the study of rates of chemical processes. Chemical kinetics includes investigations of how different experimental conditions can influence the speed of a chemical reaction and yield information about the reaction's mechanism and transition...
, permitting the control of individual action potentials at frequencies of at least up to 200 Hz (in appropriate cell types).
Even before these achievements, Roger Tsien's lab had also optimized ChR2 towards the same direction (higher steady-state conductance), by creating chimeras of ChR1 and ChR2 and mutating them at decisive amino acids, yielding the tools ChEF and ChIEF.
Finally, the groups of Hegemann and Deisseroth discovered VChR1 in the multicellular algae
Volvox carteri, and thus provided a tool - comparable to ChR2 - but with a red-shifted absorption spectrum, thus allowing excitation of two distinct cell populations at two distinct wavelengths.
Karl Deisseroth's group has also taken the lead in developing optimal viral vectors for genetic targeting of ChR2 and its derivatives, thereby pioneering many optogenetic applications in actual experiments, alone or in collaboration, including the genetically targeted remote control in rodents
in vivoIn vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
, the optogenetic induction of learning in rodents, the experimental treatment of
Parkinson's diseaseParkinson's disease is a degenerative disorder of the central nervous system...
in rats, the combination with imaging (all-optical systems) and the combination with fMRI (opto-fMRI). Other labs have pioneered essential applications of ChR2 in paradigms such as the mapping of long-range and local neural circuits, its expression from a transgenic locus - directly or in the
Cre-loxCre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice . Subsequently, the laboratory of Dr...
conditional paradigm - as well as the
two-photonTwo-photon excitation microscopy is a fluorescence imaging technique that allows imaging of living tissue up to a very high depth, that is up to about one millimeter. Being a special variant of the multiphoton fluorescence microscope, it uses red-shifted excitation light which can also excite...
excitation of ChR2, permitting the activation of individual cells.
Structure
Structurally, channelrhodopsins are
retinylidene proteinRetinylidene proteins are a family of proteins that use retinal as chromophore for light reception. Proteins of this family are also called opsins...
s. They are seven-transmembrane proteins like
rhodopsinRhodopsin, also known as visual purple, is a biological pigment of the retina that is responsible for both the formation of the photoreceptor cells and the first events in the perception of light. Rhodopsins belong to the G-protein coupled receptor family and are extremely sensitive to light,...
, and contain the light-isomerizable
chromophoreA chromophore is the part of a molecule responsible for its color. The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls...
all-
trans-
retinalRetinal, also called retinaldehyde or vitamin A aldehyde, is one of the many forms of vitamin A . Retinal is a polyene chromophore, and bound to proteins called opsins, is the chemical basis of animal vision...
(an
aldehydeAn aldehyde is an organic compound containing a formyl group. This functional group, with the structure R-CHO, consists of a carbonyl center bonded to hydrogen and an R group....
derivativeIn chemistry, a derivative is a compound that is derived from a similar compound by some chemical or physical process. In the past it was also used to mean a compound that can be imagined to arise from another compound, if one atom is replaced with another atom or group of atoms, but modern...
of
vitamin AVitamin A is a vitamin that is needed by the retina of the eye in the form of a specific metabolite, the light-absorbing molecule retinal, that is necessary for both low-light and color vision...
). The retinal chromophore is covalently linked to the rest of the protein via a protonated
Schiff baseA Schiff base, named after Hugo Schiff, is a compound with a functional group that contains a carbon-nitrogen double bond with the nitrogen atom connected to an aryl or alkyl group, not hydrogen....
. Whereas most 7-transmembrane proteins are
G protein-coupled receptorG protein-coupled receptors , also known as seven-transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, and G protein-linked receptors , comprise a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal...
s that open other ion channels indirectly via
second messengerSecond messengers are molecules that relay signals from receptors on the cell surface to target molecules inside the cell, in the cytoplasm or nucleus. They relay the signals of hormones like epinephrine , growth factors, and others, and cause some kind of change in the activity of the cell...
s (i.e. they are metabotropic), channelrhodopsins directly form ion channels (i.e. they are ionotropic). This makes cellular depolarization extremely fast, robust, and useful for bioengineering and neuroscience applications, including
photostimulationPhotostimulation is the use of light to artificially activate biological compounds, cells, or even whole organisms. Photostimulation can be used to noninvasively probe the causal relationships between different biological processes, using only light...
.
Function
ChR2 absorbs blue light with an absorption and
action spectrumAn action spectrum is the rate of a physiological activity plotted against wavelength of light. It shows which wavelength of light is most effectively used in a specific chemical reaction. Some reactants are able to use specific wavelengths of light more effectively to complete their reactions...
maximum at 480 nm. When the all-
trans-retinal complex absorbs a
photonIn physics, a photon is an elementary particle, the quantum of the electromagnetic interaction and the basic unit of light and all other forms of electromagnetic radiation. It is also the force carrier for the electromagnetic force...
, it induces a conformational change from all-
trans to 13-
cis-retinal. This conformational change introduces a further conformational change in the transmembrane protein, opening the pore to at least 6Å. Within a few milliseconds, the retinal relaxes back to the all-trans form, closing the pore and stopping the flow of ions.
Designer-Channelrhodopsins
The C-terminal end of Channelrhodopsin-2 extends into the intracellular space and can be replaced by fluorescent proteins without affecting channel function. This kind of fusion construct can be very useful to visualize the morphology of ChR2 expressing cells. Channel kinetics can be modified by point mutations close to the retinal binding pocket. For example, closing of the channel after optical activation can be substantially delayed by mutating a specific protein residue, C128. This modification results in a super-sensitive Channelrhodopsin that can be opened by a blue light pulse and closed by a green or yellow light pulse. Mutating the E123 residue accelerates channel kinetics, and the resulting ChR2 mutants have been used to spike neurons at up to 200 Hz. T159 mutants display increased photocurrents, and a combination of T159 and E123 (ET/TC) is both faster and more powerful than the original ChR2. In the future, directed molecular engineering might also be applied to develop channelrhodopsins with altered spectral sensitivity.
Applications
Channelrhodopsins can be readily expressed in excitable cells such as neurons using a variety of transfection techniques (viral
transfectionTransfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
,
electroporationElectroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
,
gene gunA gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA...
). The light absorbing pigment retinal is already present in most cells (of vertebrates) in the form of
Vitamin AVitamin A is a vitamin that is needed by the retina of the eye in the form of a specific metabolite, the light-absorbing molecule retinal, that is necessary for both low-light and color vision...
. This makes
depolarizationIn biology, depolarization is a change in a cell's membrane potential, making it more positive, or less negative. In neurons and some other cells, a large enough depolarization may result in an action potential...
of excitable cells very straightforward, useful for many bioengineering and neuroscience applications such as
photostimulationPhotostimulation is the use of light to artificially activate biological compounds, cells, or even whole organisms. Photostimulation can be used to noninvasively probe the causal relationships between different biological processes, using only light...
of neurons for probing of neural circuits. The blue-light sensitive ChR2 and the yellow light-activated chloride pump
halorhodopsinHalorhodopsin is a light-driven ion pump, specific for chloride ions, and found in phylogenetically ancient archaea, known as halobacteria...
together enable multiple-color optical activation and silencing of neural activity with millisecond precision. VChR1 form from the colonial alga
Volvox carteri absorbs maximally at 535 nm and had been used to stimulate cells with yellow light (580 nm). The emerging field of controlling networks of genetically modified cells with light has been termed
OptogeneticsOptogenetics is the combination of genetic and optical methods to control specific events in targeted cells of living tissue, even within freely moving mammals and other animals, with the temporal precision needed to keep pace with functioning intact biological systems.In 2010, optogenetics was...
.
Using fluorescently labeled ChR2, light-stimulated axons and synapses can be identified in intact brain tissue. This is useful to study the molecular events during the induction of
synaptic plasticityIn neuroscience, synaptic plasticity is the ability of the connection, or synapse, between two neurons to change in strength in response to either use or disuse of transmission over synaptic pathways. Plastic change also results from the alteration of the number of receptors located on a synapse...
. ChR2 has also been used to map long-range connections from one side of the brain to the other, and to map the spatial location of specific inputs on the dendritic tree of individual neurons.
The behavior of transgenic animals expressing ChR2 in subpopulations of neurons can be remote-controlled by intense blue light. This has been demonstrated in nematodes, fruit flies, zebrafish, and in mice. Visual function in blind mice can be partially restored by expressing ChR2 in inner retinal cells. In the future, ChR2 might also find medical applications, e.g. in certain forms of retinal degeneration or for
deep brain stimulationDeep brain stimulation is a surgical treatment involving the implantation of a medical device called a brain pacemaker, which sends electrical impulses to specific parts of the brain...
.
Further reading
(Using channelrhodopsin in transgenic mice to study brain circuitry) (Using channelrhodopsin potentially to treat blindness)
External links