Channelrhodopsin
[2] Expressed in cells of other organisms, they enable light to control electrical excitability, intracellular acidity, calcium influx, and other cellular processes (see optogenetics).
Variants that are sensitive to different colors of light or selective for specific ions (ACRs, KCRs) have been cloned from other species of algae and protists.
[4] Photocurrents of the Chlorophyceae Haematococcus pluvialis and Chlamydomonas reinhardtii were studied over many years in the groups of Oleg Sineshchekov and Peter Hegemann.
[10] Both sequences were found to function as single-component light-activated cation channels in a Xenopus oocytes and human kidney cells (HEK).
Recently discovered viral channelrhodopsins (VCR1) are localized to the membrane of the endoplasmic reticulum and lead to calcium release when illuminated.
[19] In 2005, three groups sequentially established ChR2 as a tool for genetically targeted optical remote control (optogenetics) of neurons, neural circuits and behavior.
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.
[22] 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,[23] 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.
A point mutation H134R (exchanging the amino acid Histidine in position 134 of the native protein for an Arginine) resulted in increased steady-state conductance, as described in a 2005 paper that also established ChR2 as an optogenetic tool in C. elegans.
[22] 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.
[25][26] 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 (in appropriate cell types).
[35] Other labs have pioneered the combination of ChR2 stimulation with calcium imaging for all-optical experiments,[36] mapping of long-range[37] and local[38] neural circuits, ChR2 expression from a transgenic locus – directly[39] or in the Cre-lox conditional paradigm[38] – as well as the two-photon excitation of ChR2, permitting the activation of individual cells.
[20][36] Point mutations close to the retinal binding pocket have been shown to affect the biophysical properties of the channelrhodopsin, resulting in a variety of different tools.
[25] Variants with extended open time (ChR2-XXL) produce extremely large photocurrents and are very light sensitive on the population level.
[30][47] ReaChR has improved membrane trafficking and strong expression in mammalian cells, and has been used for minimally invasive, transcranial activation of brainstem motoneurons.
After some engineering to improve membrane trafficking and speed, the resulting tool (CheRiff) produced large photocurrents at 460 nm excitation.
[59][60] Anion-conducting channelrhodopsins (iChloC, iC++, GtACR) inhibit neuronal spiking in cell culture and in intact animals when illuminated with blue light.
The light-absorbing pigment retinal is present in most cells (of vertebrates) as vitamin A, making it possible to photostimulate neurons without adding any chemical compounds.
Controlling networks of genetically modified cells with light, an emerging field known as Optogenetics., allows researchers now to explore the causal link between activity in a specific group of neurons and mental events, e.g. decision making.
