Channelrhodopsins are light-gated ion channels. They are useful molecules, enabling the use of light to control intracellular acidity, calcium influx, and electrical excitability.

Three channelrhodopsins are currently known: Channelrhodopsin-1 and Channelrhodopsin-2 are both light gated proton channels, but Channelrhodopsin-2 exhibits in addition some conductance for cations. Both proteins serve as sensory photoreceptors in the green alga Chlamydomonas controlling behavioural responses like photophobic and phototaxic responses at high light intensities. A third channelrhodopsin with red-shifted absorption (VChR1) has been discovered in the multicellular alga Volvox.^[1]

Structurally, channelrhodopsins are retinylidene proteins. They are thought to be seven-transmembrane proteins like rhodopsin, and contain the light-isomerizable vitamin A derivative all-trans-retinal. However, whereas most opsins are G-protein coupled receptors that open other ion channels indirectly via messengers, channelrhodopsins form a channel pore itself. This makes cellular depolarization extremely fast, robust, and useful for bioengineering and neuroscience applications, including photostimulation. Peak absorbance of the ChR2 retinal complex is about 460 nm (blue). Together with the yellow light-activated chloride pump halorhodopsin, which inhibits neurons, ChR2 enables multiple-color optical activation and silencing of neural activity.

Mechanics

Scheme of ChR2-RFP fusion construct

Channelrhodopsin (ChR) consists of a 7-transmembrane helix protein, as in many other rhodopsins, with a retinal chromophore that is covalently linked to the protein via a protonated Schiff base. The peak absorbance of the ChR2 retinal complex is about 470 nm (blue). When the all-trans retinal complex absorbs light, it induces a conformational change, probably 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 6A. The 13-cis-retinal naturally relaxes with time back to the all-trans-retinal which closes the pore, stopping the flow of ions.^[2] The 7-transmembrane nature of Channelrhodopsin-2 is fairly rare to ion channels which usually consist of similar repeating parts.^[2] The C-terminal end of ChR2 extends well into the intracelluar space, whereas the N-terminal end consists of the 7-transmembrane section. Without affecting channel function, the C-terminus can be replaced by red or yellow fluorescent protein (YFP) to visualize the morphology of ChR2 expressing cells. Channelrhodopsin-1 was discovered first but has found little use, because in the original experiments in Xenopus oocytes the expression level was quite low.^[3] Moreover the life time of the conducting state is three times shorter than for Channelrhodopsin-2, which results in faster kinetics but smaller stationary currents in continuous light.^[4] Channelrhodopsin-1 from from the colonial alga Volvox carteri (VChR1) comprises the most red-shifted absorption with maximum at 535 nm.^[1] It can be used in the Neuroscience for stimulation of cells with yellow light (580 nm) which is less harmful than the blue light needed for ChR2.

Applications

Channelrhodopsins can be readily expressed in excitable cells such as neurons using a variety of transfection techniques (viral transfection, electroporation, gene gun). The light absorbing pigment retinal is already present in most cells (of vertebrates) in the form of Vitamin A. This makes depolarization of excitable cells very straightforward, useful for many bioengineering and neuroscience applications such as photostimulation of neurons for probing of neural circuits. The blue-light sensitve ChR2 and the yellow light-activated chloride pump halorhodopsin together enable multiple-color optical activation and silencing of neural activity with millisecond precision.^[5] The emerging field of controlling networks of genetically modified cells with light has been termed Optogenetics.

Using fluorescently labeled ChR2, light-stimulated axons and synapses can be identified in intact brain tissue.^[6] This is useful to study the molecular events during the induction of synaptic plasticity.^[7] ChR2 has also been used to map long-range connections from one side of the brain to the other.^[8]

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.^[9][10] Visual function in blind mice can be partially restored by expressing ChR2 in bipolar cells of the retina.^[11] In the future, ChR2 might also find medical applications, e.g. in certain forms of retinal degeneration or for deep brain stimulation.

References

1. ^ ^{a b} Zhang F, Prigge M, Beyrière F, et al (April 23, 2008). "Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri". Nat. Neurosci. 11 (6): 631–3. doi:10.1038/nn.2120. PMID 18432196. 
2. ^ ^{a b} Nagel G, Szellas T, Huhn W, et al (November 25, 2003). "Channelrhodopsin-2, a directly light-gated cation-selective membrane channel". Proc. Natl. Acad. Sci. U.S.A. 100 (24): 13940–5. doi:10.1073/pnas.1936192100. PMID 14615590. 
3. ^ Nagel G, Ollig D, Fuhrmann M, et al (June 28, 2002). "Channelrhodopsin-1: a light-gated proton channel in green algae". Science 296 (5577): 2395–8. doi:10.1126/science.1072068. PMID 12089443. 
4. ^ Berthold P, Tsunoda SP, Ernst OPet al (2008). "Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization". Plant Cell 20 (6): 1665-77. PMID 18552201. 
5. ^ Zhang F, Wang LP, Brauner M, et al (April 5, 2007). "Multimodal fast optical interrogation of neural circuitry". Nature 446 (7136): 633–9. doi:10.1038/nature05744. PMID 17410168. 
6. ^ Zhang YP, Oertner TG (February 4, 2007). "Optical induction of synaptic plasticity using a light-sensitive channel". Nat. Methods 4 (2): 139–41. doi:10.1038/nmeth988. PMID 17195846. 
7. ^ Zhang YP, Holbro N, Oertner TG (August 19, 2008). "Optical induction of plasticity at single synapses reveals input-specific accumulation of αCaMKII". Proc. Natl. Acad. Sci. U.S.A. 105: 12039–44. doi:10.1073/pnas.0802940105. PMID 18697934. 
8. ^ Petreanu L, Huber D, Sobczyk A, Svoboda K (May 1, 2007). "Channelrhodopsin-2–assisted circuit mapping of long-range callosal projections". Nat. Neurosci. 10 (5): 663–8. doi:10.1038/nn1891. PMID 17435752. 
9. ^ Douglass AD, Kraves S, Deisseroth K, Schier AF, Engert F (August 5, 2008). "Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons". Current Biology 18 (15): 1133-7. PMID 18682213. 
10. ^ Huber D, Petreanu L, Ghitani N, Ranade S, Hromádka T, Mainen Z, Svoboda K (Jan 3, 2008). "Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice". Nature 451 (7174): 61-4. PMID 18094685. 
11. ^ Lagali PS, Balya D, Awatramani GB, et al (June 1, 2008). "Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration". Nat. Neurosci. 11 (6): 667–75. doi:10.1038/nn.2117. PMID 18432197. 

Further reading

• Arenkiel BR, Peca J, Davison IG, et al (April 2007). "In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2". Neuron 54 (2): 205–18. doi:10.1016/j.neuron.2007.03.005. PMID 17442243.  (Using channelrhodopsin in transgenic mice to study brain circuitry)
• Bi A, Cui J, Ma YP, et al (April 2006). "Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration". Neuron 50 (1): 23–33. doi:10.1016/j.neuron.2006.02.026. PMID 16600853.  (Using channelrhodopsin potentially to treat blindness)

External links

• Channelrhodopsin mediating optical activation of neurons
• Optogenetics Resource Center