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==Designer-Channelrhodopsins==
==Designer-Channelrhodopsins==


The [[C-terminal]] end of Channelrhodopsin-2 extends into the intracellular space and can be replaced by [[fluorescent protein]]s without affecting channel function. This kind of [[fusion construct]]s can be very useful to visualize the morphology of ChR2 expressing cells.<ref name="Boyden" /><ref name="ZhangOertner2007">{{cite journal |author=Zhang YP, Oertner TG |title=Optical induction of synaptic plasticity using a light-sensitive channel |journal=Nat. Methods |volume=4 |issue=2 |pages=139–41 |date=February 4, 2007 |pmid=17195846 |doi=10.1038/nmeth988}}</ref> 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 orange light pulse.<ref name="Berndt">{{cite journal |author=Berndt A, Yizhar O, Gunaydin LA, Hegemann P, Deisseroth K |title=Bi-stable neural state switches |journal=Nat. Neuroscience |volume=12 |issue=2 |pages=229-234 |date=December 2, 2008 |doi=10.1038/nn.2247}}</ref> In the future, directed molecular engineering might also be applied to develop channelrhodopsins with altered spectral sensitivity.
The [[C-terminal]] end of Channelrhodopsin-2 extends into the intracellular space and can be replaced by [[fluorescent protein]]s without affecting channel function. This kind of [[fusion construct]]s can be very useful to visualize the morphology of ChR2 expressing cells.<ref name="Boyden" /><ref name="ZhangOertner2007">{{cite journal |author=Zhang YP, Oertner TG |title=Optical induction of synaptic plasticity using a light-sensitive channel |journal=Nat. Methods |volume=4 |issue=2 |pages=139–41 |date=February 4, 2007 |pmid=17195846 |doi=10.1038/nmeth988}}</ref> 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.<ref name="Berndt">{{cite journal |author=Berndt A, Yizhar O, Gunaydin LA, Hegemann P, Deisseroth K |title=Bi-stable neural state switches |journal=Nat. Neuroscience |volume=12 |issue=2 |pages=229-234 |date=December 2, 2008 |doi=10.1038/nn.2247}}</ref><ref name="Schoenenberger2009">{{cite journal |author=Schoenenberger P, Gerosa D, Oertner TO |title=Temporal control of immediate early gene induction by light |journal=PLoS ONE |volume=4 |issue=12 |pages=e8185 |date=December 4, 2009 |doi=10.1371/journal.pone.0008185}}</ref> In the future, directed molecular engineering might also be applied to develop channelrhodopsins with altered spectral sensitivity.


==Applications==
==Applications==

Revision as of 19:15, 5 December 2009

Channelrhodopsins are a subfamily of opsin proteins that function as light-gated ion channels.[1] They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis, i.e. movement in response to light. Expressed in cells of other organisms, they enable the use of light to control intracellular acidity, calcium influx, electrical excitability, and other cellular processes. Three channelrhodopsins are currently known: Channelrhodopsin-1 (ChR1), Channelrhodopsin-2 (ChR2), and Volvox Channelrhodopsin (VChR1). All known Channelrhodopsins are unspecific cation channels, conducting H+, Na+, K+, and Ca2+ ions.

History

Channelrhodopsin-1 was discovered to be a light-activated ion channel in the green algae Chlamydomonas reinhardtii by Georg Nagel and colleagues in 2002.[2] Channelrhodopsin-2, which was also isolated from Chlamydomonas reinhardtii, was applied in neurobiology by Karl Deisseroth and colleagues in 2005, and it has found widespread use in neuroscience[1][3] since then. Compared to ChR1, the life time of the conducting state of ChR2 is two times longer. This results in larger stationary currents, but slower kinetics.[4]

VChR1, which has red-shifted absorption, was discovered in the multicellular alga Volvox.[5]

Structure

Structurally, channelrhodopsins are retinylidene proteins. They are seven-transmembrane proteins like rhodopsin, and contain the light-isomerizable chromophore all-trans-retinal (an aldehyde derivative of vitamin A). The retinal chromophore is covalently linked to the rest of the protein via a protonated Schiff base. Whereas most 7-transmembrane proteins are G protein-coupled receptors that open other ion channels indirectly via second messengers (i.e. they are metabotropic), channelrhodopsins directly form ion channels (i.e. they are ionotropic).[1] This makes cellular depolarization extremely fast, robust, and useful for bioengineering and neuroscience applications, including photostimulation.

Function

Scheme of ChR2-RFP fusion construct

ChR2 absorbs blue light with an absorption maximum at 480 nm.[6] When the all-trans-retinal complex absorbs a photon, 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.[1]

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 constructs can be very useful to visualize the morphology of ChR2 expressing cells.[3][7] 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.[8][9] 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 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.[3] The blue-light sensitive ChR2 and the yellow light-activated chloride pump halorhodopsin together enable multiple-color optical activation and silencing of neural activity with millisecond precision.[10][11] 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).[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.[7] This is useful to study the molecular events during the induction of synaptic plasticity.[12] 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. [13][14]

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.[15][16] Visual function in blind mice can be partially restored by expressing ChR2 in bipolar cells of the retina.[17] 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 c d 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. PMC 283525. PMID 14615590. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)
  2. ^ 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. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)
  3. ^ a b c Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005). "Millisecond-timescale, genetically-targeted optical control of neural activity". Nature Neuroscience. 8 (9): 1263–1268. doi:10.1038/nn1525.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  4. ^ Berthold P, Tsunoda SP, Ernst OP; et al. (2008). "Channelrhodopsin-1 initiates phototaxis and photophobic responses in chlamydomonas by immediate light-induced depolarization". Plant Cell. 20 (6): 1665–77. doi:10.1105/tpc.108.057919. PMID 18552201. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)
  5. ^ 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. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)
  6. ^ Christian Bamann, Taryn Kirsch, Georg Nagel, Ernst Bamberg (2008). "Spectral Characteristics of the Photocycle of Channelrhodopsin-2 and Its Implication for Channel Function". J Mol Biol. 375 (3): 686–694. doi:10.1016/j.jmb.2007.10.072. {{cite journal}}: Cite has empty unknown parameter: |unused_data= (help); Text "pmid:18037436" ignored (help)CS1 maint: multiple names: authors list (link)
  7. ^ a b 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.
  8. ^ Berndt A, Yizhar O, Gunaydin LA, Hegemann P, Deisseroth K (December 2, 2008). "Bi-stable neural state switches". Nat. Neuroscience. 12 (2): 229–234. doi:10.1038/nn.2247.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  9. ^ Schoenenberger P, Gerosa D, Oertner TO (December 4, 2009). "Temporal control of immediate early gene induction by light". PLoS ONE. 4 (12): e8185. doi:10.1371/journal.pone.0008185.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
  10. ^ Han X, Boyden ES (March 21, 2007). "Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity, with Single-Spike Temporal Resolution". PLoS ONE. 2 (3): e299. doi:10.1371/journal.pone.0000299. PMID 17375185.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  11. ^ 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. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)
  12. ^ 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  13. ^ 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.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  14. ^ Petreanu L, Mao Y, Sternson SM, Svoboda K (Feb 26, 2009). "The subcellular organization of neocortical excitatory connections". Nature. 457 (7233): 1142–5. doi:10.1038/nature07709. PMID 19151697.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ^ 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. doi:10.1016/j.cub.2008.06.077. PMID 18682213.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  16. ^ Huber D, Petreanu L, Ghitani N, Ranade S, Hromádka T, Mainen Z, Svoboda K (January 3, 2008). "Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice". Nature. 451 (7174): 61–4. doi:10.1038/nature06445. PMID 18094685.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ^ 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. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)

Further reading

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