Channelrhodopsin

from Wikipedia, the free encyclopedia
Channelrhodopsin
Identifier
Gene name (s) ChR1, ChR2, VChR1
Transporter classification
TCDB 3.E.1.7
designation Ion-shifting microbial rhodopsin
Occurrence
Parent taxon Seaweed

Channelrhodopsins (German also: Kanalrhodopsine ) are ion channels that occur in the cell membrane of certain single-cell algae. Blue light leads to the opening of these channels (English light-gated ) and the influx of ions into the cell. Channelrhodopsins make the membrane potential and the ion concentration in the cytosol dependent on the light intensity. If a gene for channelrhodopsin is introduced into nerve cells, the electrical excitability of these nerve cells can be controlled by light pulses ( optogenetics ).

The first channelrhodopsins to be discovered, channelrhodopsin-1 ( ChR1 ) and channelrhodopsin-2 ( ChR2 ), serve green algae of the genus Chlamydomonas as sensory photoreceptors . They conduct positively charged ions ( cations ) into the cell and thus control negative and positive phototaxic reactions when there is a high incidence of light. VChR1 was found in the multicellular alga Volvox ; its absorption maximum is at a higher wavelength than ChR1 and ChR2. However, it shows an 80% match of the amino acid sequence to the ChR1 group, which means that these proteins are considered to be homologous to one another. In addition to these cation-conducting channelrhodopsins, channelrhodopsins were found in cryptophyte algae , which conduct negatively charged ions (anions). If anion-conducting channelrhodopsins ( ACR ) are smuggled into nerve cells, these nerve cells can be prevented from being activated by lighting ( silencing ).

Layout and function

Channelrhodopsins (ChR), like other rhodopsins , are proteins with seven helical transmembrane domains and a retinal chromophore, which is covalently bound to the protein as a protonated Schiff base . The absorption maximum of ChR2 is around 460–470  nm in the blue. As soon as the all-trans-retinal in the protein-retinal complex absorbs light, it isomerizes to a 13-cis-retinal and thereby causes a conformational change of the protein. This leads to the opening of the pore in the protein, its diameter is at least 0.6 nm. After some time, the 13-cis retinal relaxes back to the all-trans retinal, whereby the pore closes again and the ion flow is interrupted. While most G-protein-coupled receptors (including rhodopsin) open ion channels indirectly by means of secondary messenger substances , in the case of channelrhodopsins the protein itself forms a pore. This structure enables a very fast and reliable depolarization of the cell. In the case of heterologous expression of ChR2 in nerve cells , a short light pulse (1–2 ms) can trigger an action potential . Most cell types have sufficient retinal (vitamin A) to enable the production of functional channelrhodopsins without the addition of retinal.

Variants for use in optogenic experiments

The exchange of amino acids near the retinal binding pocket ( point mutation ) influences the biophysical properties of channelrhodopsin. Various working groups have generated a large number of optogenetic tools through targeted mutations.

kinetics

The closing of the channel after optical activation can be delayed significantly by mutating protein residues C128 or D156. This modification leads to highly sensitive channel rhodopsins, which can be opened by a blue light pulse and closed by a green or yellow light pulse (step-function opsins). The mutation of the E123 residue accelerates channel kinetics (ChETA), and the resulting ChR2 mutants were used to make neurons fire at up to 200 Hz. In general, slow kinetic channelrhodopsins are more sensitive to light at the population level because open channels accumulate over time, even at low light levels.

Photocurrent amplitude

H134R and T159C mutants show increased photocurrents; a combination of T159 and E123 (ET / TC) has slightly larger photocurrents and slightly faster kinetics than wild-type ChR2. Among ChR variants, ChIEF, a chimera and point mutant of ChR1 and ChR2, shows the largest photocurrents and the least desensitization and has kinetics that are similar to ChR2.

Excitation wavelength

Chimeric channelrhodopsins were developed by combining transmembrane helices from ChR1 and VChR1, resulting in ChRs with red spectral shifts (e.g. C1V1, ReaChR). ReaChR can be incorporated into the cell membrane of mammalian cells at a very high density and has been used for minimally invasive, transcranial activation of brainstem motor neurons. The search for homologous sequences in other organisms led to spectrally improved and more red-shifted channelrhodopsins (e.g. ChrimsonR). In combination with ChR2, these yellow / red light-sensitive channelrhodopsins enable two populations of neurons to be controlled independently of one another with light pulses of different colors. A blue-shifted channelrhodopsin was discovered in the alga Scherffelia dubia . After a few mutations to improve membrane trade and speed, the resulting tool (CheRiff) resulted in large photocurrents when excited by blue light (460 nm).

Ion selectivity

The L132C mutation (CatCh) increases the permeability for calcium ions and creates very large currents. The mutation of E90 to the positively charged amino acid arginine transforms channelrhodopsin from a non-specific cation channel into a chloride-conducting channel (ChloC). The selectivity for Cl- was further improved by replacing negatively charged residues in the pore of the channel. Selective chloride-conducting channelrhodopsins (iChloC, GtACR) are used to specifically dampen the activity of certain neurons in the fruit fly and in the mouse and thus to investigate the function of these neurons for a certain behavior.

Applications in research

Schematic representation of a ChR2-RFP fusion protein. RFP is a red fluorescent protein that, like GFP , can be used to mark cell structures.

While the N-terminus includes the seven transmembrane domains, the C-terminal end of the ChR2 protein extends into the intracellular space and can be replaced or changed without the protein's function as an ion channel being impaired. Channelrhodopsins can be expressed (produced) in excitable cells such as neurons using a number of transfection techniques (viral transfection , electroporation , gene gun ) . Vitamin A, the precursor of the light-absorbing retinal chromophore, is usually already present in vertebrate cells, so that excitable cells that express a channelrhodopsin can be depolarized simply by lighting.

Because of these properties, biotechnology and neurosciences are interested in the use of channelrhodopsins, for example for applications such as the photostimulation of neurons. The blue-sensitive ChR2 in combination with the halorhodopsin chloride pump , which can be activated by yellow light, enables neuronal activity to be switched on and off within milliseconds. The emerging field of light control of genetically modified cells is known as optogenetics .

If ChR2 is marked with a fluorescent label, axons and synapses excited by light can be identified in intact brain tissue. This technique can be used to elucidate the molecular events during the induction of synaptic plasticity . With the help of ChR2, far-reaching neural pathways in the brain were mapped.

It has already been shown for nematodes , fruit flies , zebrafish and mice that the behavior of transgenic animals that express ChR2 in some of their neurons can be controlled without contact by intensive lighting with blue light .

The visual function of blind mice could be partially restored by expressing ChR2 in bipolar cells of the retina in the eye . A future medical use of ChR2 is also conceivable for certain forms of retinal degeneration or for stimulating deep-lying brain areas.

Individual evidence

  1. ↑ Georg Nagel, Doris Ollig, Markus Fuhrmann, Suneel Kateriya, Anna Maria Musti: Channelrhodopsin-1: A Light-Gated Proton Channel in Green Algae . In: Science . tape 296 , no. 5577 , June 28, 2002, ISSN  0036-8075 , p. 2395–2398 , doi : 10.1126 / science.1072068 , PMID 12089443 ( sciencemag.org [accessed December 28, 2017]).
  2. ↑ Zhang F, Prigge M, Beyrière F, et al : Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri . In: Nat. Neurosci. . 11, No. 6, April 23, 2008, pp. 631-3. doi : 10.1038 / nn.2120 . PMID 18432196 .
  3. ↑ Elena G. Govorunova, Oleg A. Sineshchekov, Roger Janz, Xiaoqin Liu, John L. Spudich: Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics . In: Science . tape 349 , no. 6248 , August 7, 2015, ISSN  0036-8075 , p. 647-650 , doi : 10.1126 / science.aaa7484 , PMID 26113638 ( sciencemag.org [accessed December 28, 2017]).
  4. ↑ Nagel G, Szellas T, Huhn W, et al : Channelrhodopsin-2, a directly light-gated cation-selective membrane channel . In: Proc. Natl. Acad. Sci. USA . 100, No. 24, November 25, 2003, pp. 13940-5. doi : 10.1073 / pnas.1936192100 . PMID 14615590 . PMC 283525 (free full text).
  5. ↑ André Berndt, Ofer Yizhar, Lisa A Gunaydin, Peter Hegemann, Karl Deisseroth: Bi-stable neural state switches . In: Nature Neuroscience . tape 12 , no. 2 , p. 229-234 .
  6. ↑ Lisa A Gunaydin, Ofer Yizhar, André Berndt, Vikaas S Sohal, Karl Deisseroth: Ultrafast opto genetic control . In: Nature Neuroscience . tape 13 , no. 3 , p. 387-392 .
  7. ↑ André Berndt, Philipp Schoenenberger, Joanna Mattis, Kay M. Tye, Karl Deisseroth: High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels . In: Proceedings of the National Academy of Sciences . tape 108 , no. 18 , May 3, 2011, ISSN  1091-6490 , p. 7595-7600 , doi : 10.1073 / pnas.1017210108 , PMID 21504945 , PMC 3088623 (free full text).
  8. ↑ John Y. Lin: A user's guide to channel rhodopsin variants: features, limitations and future developments . In: Experimental Physiology . tape 96 , no. 1 , January 1, 2011, ISSN  1469-445X , p. 19-25 , doi : 10.1113 / expphysiol.2009.051961 , PMID 20621963 , PMC 2995811 (free full text).
  9. ↑ John Y Lin, Per Magne Knutsen, Arnaud Muller, David Kleinfeld, Roger Y Tsien: ReaChR: a red-shifted variant of channelrhodopsin enables deep transcranial optogenetic excitation . In: Nature Neuroscience . tape 16 , no. 10 , September 2013, p. 1499–1508 , doi : 10.1038 / nn.3502 , PMID 23995068 , PMC 3793847 (free full text).
  10. ↑ Nathan C. Klapötke, Yasunobu Murata, Sung Soo Kim, Stefan R. powder Amanda Birdsey-Benson: Independent optical excitation of distinct neural populations . In: Nature Methods . tape 11 , no. 3 , March 1, 2014, ISSN  1548-7105 , p. 338-346 , doi : 10.1038 / nmeth.2836 , PMID 24509633 , PMC 3943671 (free full text).
  11. ↑ Sonja Kleinlogel, Katrin Feldbauer, Robert E Dempski, Heike Fotis, Phillip G Wood: Ultra light-sensitive and fast neuronal activation with the Ca2 + -permeable channelrhodopsin CatCh . In: Nature Neuroscience . tape 14 , no. 4 , March 13, 2011, p. 513-518 , doi : 10.1038 / nn.2776 .
  12. ↑ Jonas Wietek, J. Simon Wiegert, Nona Adeishvili, Franziska Schneider, Hiroshi Watanabe: Conversion of Channelrhodopsin into a Light-Gated Chloride Channel . In: Science . tape 344 , no. 6182 , April 25, 2014, ISSN  0036-8075 , p. 409-412 , doi : 10.1126 / science.1249375 , PMID 24674867 ( sciencemag.org [accessed January 16, 2017]).
  13. ↑ Jonas Wietek, Riccardo Beltramo, Massimo Scanziani, Peter Hegemann, Thomas G. Oertner: An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo . In: Scientific Reports . tape 5 , October 7, 2015, ISSN  2045-2322 , doi : 10.1038 / srep14807 , PMID 26443033 , PMC 4595828 (free full text).
  14. ^ Naoya Takahashi, Thomas G. Oertner, Peter Hegemann, Matthew E. Larkum: Active cortical dendrites modulate perception . In: Science (New York, NY) . tape 354 , no. 6319 , December 23, 2016, ISSN  1095-9203 , p. 1587-1590 , doi : 10.1126 / science.aah6066 , PMID 28008068 .
  15. ↑ Zhang F, Wang LP, Brauner M, et al : Multimodal fast optical interrogation of neural circuitry . In: Nature . 446, No. 7136, April 5, 2007, pp. 633-9. doi : 10.1038 / nature05744 . PMID 17410168 .
  16. ↑ Zhang YP, Oertner TG: Optical induction of synaptic plasticity using a light-sensitive channel . In: Nat. Methods . 4, No. 2, February 4, 2007, pp. 139-41. doi : 10.1038 / nmeth988 . PMID 17195846 .
  17. ↑ Zhang YP, Holbro N, Oertner TG: Optical induction of plasticity at single synapses reveals input-specific accumulation of αCaMKII . In: Proc. Natl. Acad. Sci. USA . 105, Aug 19, 2008, pp. 12039-44. doi : 10.1073 / pnas.0802940105 . PMID 18697934 .
  18. ↑ Petreanu L, Huber D, Sobczyk A, Svoboda K: Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections . In: Nat. Neurosci. . 10, No. 5, May 1, 2007, pp. 663-8. doi : 10.1038 / nn1891 . PMID 17435752 .
  19. ↑ Douglass AD, Kraves S, Deisseroth K , Schier AF, Engert F: Escape behavior elicited by single, channelrhodopsin-2-evoked spikes in zebrafish somatosensory neurons . In: Current Biology . 18, No. 15, August 5, 2008, pp. 1133-1137. PMID 18682213 .
  20. ↑ Huber D, Petreanu L, Ghitani N, Ranade S, Hromádka T, Mainen Z, Svoboda K: Sparse optical microstimulation in barrel cortex drives learned behavior in freely moving mice . In: Nature . 451, No. 7174, Jan. 3, 2008, pp. 61-64. PMID 18094685 .
  21. ↑ Lagali PS, Balya D, Awatramani GB, et al : Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration . In: Nat. Neurosci. . 11, No. 6, June 1, 2008, pp. 667-75. doi : 10.1038 / nn.2117 . PMID 18432197 .

literature

  • Arenkiel BR, Peca J, Davison IG, et al : In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2 . In: Neuron . 54, No. 2, April 2007, pp. 205-18. doi : 10.1016 / j.neuron.2007.03.005 . PMID 17442243 . (Use of channelrhodopsin in transgenic mice to research the neural circuitry in the brain)
  • Bi A, Cui J, Ma YP, et al : Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration . In: Neuron . 50, No. 1, April 2006, pp. 23-33. doi : 10.1016 / j.neuron.2006.02.026 . PMID 16600853 . PMC 1459045 (free full text). (Channelrhodopsin as a possible treatment for blindness)

Web links