Channelrhodopsin
Channelrhodopsins are a subfamily of retinylidene proteins that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light. Expressed in cells of other organisms, they enable light to control electrical excitability, intracellular acidity, calcium influx, and other cellular processes. Channelrhodopsin-1 and Channelrhodopsin-2 from the model organism Chlamydomonas reinhardtii are the first discovered channelrhodopsins. Variants that are sensitive to different colors of light or selective for specific ions have been cloned from other species of algae and protists.
History
and photoorientation of microalgae have been studied over more than a hundred years in many laboratories worldwide. In 1980, Ken Foster developed the first consistent theory about the functionality of algal eyes. He also analyzed published action spectra and complemented blind cells with retinal and retinal analogues, which led to the conclusion that the photoreceptor for motility responses in Chlorophyceae is rhodopsin.Photocurrents of the Chlorophyceae Haematococcus pluvialis and Chlamydomonas reinhardtii were studied over many years in the groups of Oleg Sineshchekov and Peter Hegemann. Based on action spectroscopy and simultaneous recordings of photocurrents and flagellar beating, it was determined that the photoreceptor currents and subsequent flagellar movements are mediated by rhodopsin and control phototaxis and photophobic responses. The extremely fast rise of the photoreceptor current after a brief light flash led to the conclusion that the rhodopsin and the channel are intimately linked in a protein complex, or even within one single protein.
The name "channelrhodopsin" was coined to highlight this unusual property, and the sequences were renamed accordingly. The nucleotide sequences of the rhodopsins now called channelrhodopsins ChR1 and ChR2 were finally uncovered in a large-scale EST sequencing project in C. reinhardtii. Independent submission of the same sequences to GenBank by three research groups generated confusion about their naming: The names cop-3 and cop-4 were used for initial submission by Hegemann's group; csoA and csoB by Spudich's group; and acop-1 and acop-2 by Takahashi's group. Both sequences were found to function as single-component light-activated cation channels in a Xenopus oocytes and human kidney cells.
Their roles in generation of photoreceptor currents in algal cells were characterized by Oleg Sineshchekov, Kwang-Hwan Jung and John Spudich, and Peter Berthold and Peter Hegemann.
Structure
In terms of structure, channelrhodopsins are retinylidene proteins. They are seven-transmembrane proteins like rhodopsin, and contain the light-isomerizable chromophore all-trans-retinal. The retinal chromophore is covalently linked to the rest of the protein through a protonated Schiff base. Whereas most 7-transmembrane proteins are G protein-coupled receptors that open other ion channels indirectly via second messengers, channelrhodopsins directly form ion channels. This makes cellular depolarization extremely fast, robust, and useful for bioengineering and neuroscience applications, including photostimulation.Function
The natural ChR2 absorbs blue light with an absorption and action spectrum maximum at 480 nm. When the all-trans-retinal complex absorbs a photon, it induces a conformational change from all-trans to 13-cis-retinal. This change introduces a further one in the transmembrane protein, opening the pore to at least 6 Å. Within milliseconds, the retinal relaxes back to the all-trans form, closing the pore and stopping the flow of ions. Most natural channelrhodopsins are nonspecific cation channels, conducting H+, Na+, K+, and Ca2+ ions. Anion-conducting channelrhodopsins and potassium-selective channelrhodopsins were structurally analyzed to understand their ion selectivity. ACRs and KCRs have been used to inhibit neuronal activity. Recently discovered viral channelrhodopsins are localized to the membrane of the endoplasmic reticulum and lead to calcium release when illuminated.Development as a molecular tool
In 2005, three groups sequentially established ChR2 as a tool for genetically targeted optical remote control of neurons, neural circuits and behavior.At first, Karl Deisseroth's lab demonstrated that ChR2 could be deployed to control mammalian neurons in vitro, achieving temporal precision on the order of milliseconds. Because all opsins require retinal as the light-sensing co-factor and it was unclear whether central mammalian nerve cells would contain sufficient retinal levels, but they do. It also showed, despite the small single-channel conductance, sufficient potency to drive mammalian neurons above action potential threshold. From this, channelrhodopsin became the first optogenetic tool, with which neural activity could be controlled with the temporal precision at which neurons operate. A second study was published later confirming the ability of ChR2 to control the activity of vertebrate neurons, at this time in the chick spinal cord. This study was the first wherein ChR2 was expressed alongside an optical silencer, vertebrate rhodopsin-4 in this case, demonstrating for the first time that excitable cells could be activated and silenced using these two tools simultaneously, illuminating the tissue at different wavelengths.
It was demonstrated that ChR2, if expressed in specific neurons or muscle cells, can evoke predictable behaviors, i.e. can control the nervous system of an intact animal, in this case the invertebrate C. elegans. This was the first using ChR2 to steer the behavior of an animal in an optogenetic experiment, rendering a genetically specified cell type subject to optical remote control. Although both aspects had been illustrated earlier that year by the group of Gero Miesenböck, 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 principal downsides – the small single-channel conductance, the limitation to one optimal excitation wavelength as well as the relatively long recovery time, not permitting controlled firing of neurons above 20–40 Hz – ChR2 has been optimized using genetic engineering. A point mutation H134R resulted in increased steady-state conductance, as described in a 2005 paper that also established ChR2 as an optogenetic tool in C. elegans. In 2009, Roger Tsien's lab optimized ChR2 for further increases in steady-state conductance and dramatically reduced desensitization by creating chimeras of ChR1 and ChR2 and mutating specific amino acids, yielding ChEF and ChIEF, which allowed the driving of trains of action potentials up to 100 Hz. In 2010, the groups of Hegemann and Deisseroth introduced an E123T mutation into native ChR2, yielding ChETA, which has faster on- and off-kinetics, permitting the control of individual action potentials at frequencies up to 200 Hz.
The groups of Hegemann and Deisseroth also discovered that the introduction of the point mutation C128S makes the resulting ChR2-derivative a step-function tool: Once "switched on" by blue light, ChR2 stays in the open state until it is switched off by yellow light – a modification that deteriorates temporal precision, but increases light sensitivity by two orders of magnitude. They also discovered and characterized VChR1 in the multicellular algae Volvox carteri. VChR1 produces only tiny photocurrents, but with an absorption spectrum that is red-shifted relative to ChR2. Using parts of the ChR1 sequence, photocurrent amplitude was later improved to allow excitation of two neuronal populations at two distinct wavelengths.
Deisseroth's group has pioneered many applications in live animals such as genetically targeted remote control in rodents in vivo, the optogenetic induction of learning in rodents, the experimental treatment of Parkinson's disease in rats, and the combination with fMRI. Other labs have pioneered the combination of ChR2 stimulation with calcium imaging for all-optical experiments, mapping of long-range and local neural circuits, ChR2 expression from a transgenic locus – directly or in the Cre-lox conditional paradigm – as well as the two-photon excitation of ChR2, permitting the activation of individual cells.
In March 2013, the Brain Prize was jointly awarded to Bamberg, Boyden, Deisseroth, Hegemann, Miesenböck, and Nagel for "their invention and refinement of optogenetics". The same year, Hegemann and Nagel received the Louis-Jeantet Prize for Medicine for "the discovery of channelrhodopsin". In 2015, Boyden and Deisseroth received the Breakthrough Prize in Life Sciences and in 2020, Miesenböck, Hegemann and Nagel received the for the development of optogenetics.