Anion-conducting channelrhodopsin

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Figure 1: It took 5 point mutations to create iChloC from cation-conducting Channelrhodopsin-2. ChloC Scheme 2.gif
Figure 1: It took 5 point mutations to create iChloC from cation-conducting Channelrhodopsin-2.

Anion-conducting channelrhodopsins are light-gated ion channels that open in response to light and let negatively charged ions (such as chloride) enter a cell. All channelrhodopsins use retinal as light-sensitive pigment, but they differ in their ion selectivity. Anion-conducting channelrhodopsins are used as tools to manipulate brain activity in mice, fruit flies and other model organisms (Optogenetics). Neurons expressing anion-conducting channelrhodopsins are silenced when illuminated with light, an effect that has been used to investigate information processing in the brain. For example, suppressing dendritic calcium spikes in specific neurons with light reduced the ability of mice to perceive a light touch to a whisker. [2] Studying how the behavior of an animal changes when specific neurons are silenced allows scientists to determine the role of these neurons in the complex circuits controlling behavior.

Contents

The first anion-conducting channelrhodopsins were engineered from the cation-conducting light-gated channel Channelrhodopsin-2 by removing negatively charged amino acids from the channel pore (Fig. 1). [3] As the main anion of extracellular fluid is chloride (Cl−), anion-conducting channelrhodopsins are also known as "chloride-conducting channelrhodopsins" (ChloCs). Naturally occurring anion-conducting channelrhodopsins (ACRs) were subsequently identified in cryptophyte algae. [4] [5] [6] The crystal structure of the natural GtACR1 has recently been solved, paving the way for further protein engineering. [7] [8]

Structure of bromide-bound GtACR1 (PDB: 7LE1). The two gray planes indicate the hydrocarbon boundaries of the lipid bilayer and were calculated with the ANVIL algorithm. 7LE1.gif
Structure of bromide-bound GtACR1 (PDB: 7LE1). The two gray planes indicate the hydrocarbon boundaries of the lipid bilayer and were calculated with the ANVIL algorithm.

Variants

namespecies of originabsorptionreferenceproperties, applications
slowChloC Chlamydomonas reinhardtii blueWietek et al. 2014 [3] first generation, mixed conductance
iC1C2 Chlamydomonas reinhardtii blueBerndt et al. 2014 [10] first generation, mixed conductance
iChloC Chlamydomonas reinhardtii blueWietek et al. 2015 [1] inhibition of perception in mice [2]
iC++ Chlamydomonas reinhardtii blueBerndt et al. 2016 [11] inhibition of sleep in mice [12]
GtACR1 Guillardia theta greenGovorunova et al. 2015 [4] inhibition of behavior in Drosophila [13] [14] inhibition of rat heart muscle cells [15] holographic spike suppression in mouse cortex [16]
GtACR1(C102A) Guillardia theta green on

red off

Govorunova et al. 2018 [6] bistable
GtACR1(R83Q/N239Q) FLASH Guillardia theta green onKato et al. 2018 [7] very fast closing, large currents

inhibition of swimming in C. elegans, inhibition of spiking in mouse [7]

GtACR2 Guillardia theta blueGovorunova et al. 2015 [4] inhibition of behavior in Drosophila [13] inhibition of fear extinction in mice [17]
PsACR1 Proteomonas sulcata greenWietek et al. 2016, [18] Govorunova et al. 2016 [19] large currents
ZipACR Proteomonas sulcata greenGovorunova et al. 2017 [5] very fast
RapACR Rhodomonas salina greenGovorunova et al. 2018 [6] very fast, large currents
SwiChR++ Chlamydomonas reinhardtii blue on

red off

Berndt et al. 2016 [11] bistable
Phobos CA Chlamydomonas reinhardtii blue on

red off

Wietek et al. 2017 [20] bistable
Aurora Chlamydomonas reinhardtii orange-redWietek et al. 2017 [20] stop locomotion of Drosophila larvae
MerMAIDsunknowngreenOppermann et al. 2019 [21] rapidly inactivating

Applications

Anion-conducting channelrhodopsins (ACRs) have been used as optogenetic tools to inhibit neuronal activation. When expressed in nerve cells, ACRs act as light-gated chloride channels. Their effect on the activity of the neuron is comparable to GABAA receptors, ligand-gated chloride channels found in inhibitory synapses: As the chloride concentration in mature neurons is very low, illumination results in an inward flux of negatively charged ions, clamping the neuron at the chloride reversal potential (- 65 mV). Under these conditions, excitatory synaptic inputs are not able to efficiently depolarize the neuron. This effect is known as shunting inhibition (as opposed to inhibition by hyperpolarization). Illuminating the dendrite prevents the generation of dendritic calcium spikes while illumination of the entire neuron blocks action potential initiation in response to sensory stimulation. [2] [1] Axon terminals, however, have a higher chloride concentration and are therefore excited by ACRs. [22] To inhibit neurons with wide-field illumination, it has proven useful to restrict ACRs to the somatic compartment (ST variants). [17] [16]

Due to their high light sensitivity, ACRs can be activated with dim light which does not interfere with visual stimulation, even in very small animals like the fruit fly Drosophila . [14] When combined with a red-light sensitive cation-conducting channelrhodopsin, ACRs allow for bidirectional control of neurons: Silencing with blue light, activation with red light ('Bipoles'). [23]

Further reading

Neuron Review (2017): Silencing neurons: Tools, Applications, and Experimental Constraints [24]

Research highlight: A better way to turn off neurons [25]

Perspective: Expanding the optogenetics toolkit [26]

Related: Halorhodopsin, a light-driven chloride pump

References

  1. 1 2 3 Wietek, Jonas; Beltramo, Riccardo; Scanziani, Massimo; Hegemann, Peter; Oertner, Thomas G.; Wiegert, J. Simon (2015-10-07). "An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo". Scientific Reports. 5 14807. Bibcode:2015NatSR...514807W. doi:10.1038/srep14807. ISSN   2045-2322. PMC   4595828 . PMID   26443033.
  2. 1 2 3 Takahashi, Naoya; Oertner, Thomas G.; Hegemann, Peter; Larkum, Matthew E. (2016-12-23). "Active cortical dendrites modulate perception". Science. 354 (6319): 1587–1590. Bibcode:2016Sci...354.1587T. doi:10.1126/science.aah6066. ISSN   0036-8075. PMID   28008068. S2CID   28317052.
  3. 1 2 Wietek, Jonas; Wiegert, J. Simon; Adeishvili, Nona; Schneider, Franziska; Watanabe, Hiroshi; Tsunoda, Satoshi P.; Vogt, Arend; Elstner, Marcus; Oertner, Thomas G.; Hegemann, Peter (2014-04-25). "Conversion of Channelrhodopsin into a Light-Gated Chloride Channel". Science. 344 (6182): 409–412. Bibcode:2014Sci...344..409W. doi: 10.1126/science.1249375 . ISSN   0036-8075. PMID   24674867. S2CID   206554245.
  4. 1 2 3 Govorunova, Elena G.; Sineshchekov, Oleg A.; Janz, Roger; Liu, Xiaoqin; Spudich, John L. (2015-08-07). "Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics". Science. 349 (6248): 647–650. Bibcode:2015Sci...349..647G. doi:10.1126/science.aaa7484. ISSN   0036-8075. PMC   4764398 . PMID   26113638.
  5. 1 2 Govorunova, Elena G.; Sineshchekov, Oleg A.; Rodarte, Elsa M.; Janz, Roger; Morelle, Olivier; Melkonian, Michael; Wong, Gane K.-S.; Spudich, John L. (2017-03-03). "The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity". Scientific Reports. 7 43358. Bibcode:2017NatSR...743358G. doi:10.1038/srep43358. ISSN   2045-2322. PMC   5335703 . PMID   28256618.
  6. 1 2 3 Govorunova, Elena G.; Sineshchekov, Oleg A.; Hemmati, Raheleh; Janz, Roger; Morelle, Olivier; Melkonian, Michael; Wong, Gane K.-S.; Spudich, John L. (2018-05-01). "Extending the Time Domain of Neuronal Silencing with Cryptophyte Anion Channelrhodopsins". eNeuro. 5 (3): ENEURO.0174–18.2018. doi:10.1523/ENEURO.0174-18.2018. ISSN   2373-2822. PMC   6051594 . PMID   30027111.
  7. 1 2 3 Kato, Hideaki E.; Kim, Yoon Seok; Paggi, Joseph M.; Evans, Kathryn E.; Allen, William E.; Richardson, Claire; Inoue, Keiichi; Ito, Shota; Ramakrishnan, Charu (2018-08-29). "Structural mechanisms of selectivity and gating in anion channelrhodopsins". Nature. 561 (7723): 349–354. Bibcode:2018Natur.561..349K. doi:10.1038/s41586-018-0504-5. ISSN   0028-0836. PMC   6317992 . PMID   30158697.
  8. ↑ Kim, Yoon Seok; Kato, Hideaki E.; Yamashita, Keitaro; Ito, Shota; Inoue, Keiichi; Ramakrishnan, Charu; Fenno, Lief E.; Evans, Kathryn E.; Paggi, Joseph M. (2018-08-29). "Crystal structure of the natural anion-conducting channelrhodopsin GtACR1". Nature. 561 (7723): 343–348. Bibcode:2018Natur.561..343K. doi:10.1038/s41586-018-0511-6. ISSN   0028-0836. PMC   6340299 . PMID   30158696.
  9. ↑ Postic, Guillaume; Ghouzam, Yassine; Guiraud, Vincent; Gelly, Jean-Christophe (2016). "Membrane positioning for high- and low-resolution protein structures through a binary classification approach". Protein Engineering, Design and Selection. 29 (3): 87–91. doi: 10.1093/protein/gzv063 . PMID   26685702.
  10. ↑ Berndt, Andre; Lee, Soo Yeun; Ramakrishnan, Charu; Deisseroth, Karl (2014-04-25). "Structure-Guided Transformation of Channelrhodopsin into a Light-Activated Chloride Channel". Science. 344 (6182): 420–424. Bibcode:2014Sci...344..420B. doi:10.1126/science.1252367. ISSN   0036-8075. PMC   4096039 . PMID   24763591.
  11. 1 2 Berndt, Andre; Lee, Soo Yeun; Wietek, Jonas; Ramakrishnan, Charu; Steinberg, Elizabeth E.; Rashid, Asim J.; Kim, Hoseok; Park, Sungmo; Santoro, Adam (2016-01-26). "Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity". Proceedings of the National Academy of Sciences. 113 (4): 822–829. Bibcode:2016PNAS..113..822B. doi: 10.1073/pnas.1523341113 . ISSN   0027-8424. PMC   4743797 . PMID   26699459.
  12. ↑ Chung, Shinjae; Weber, Franz; Zhong, Peng; Tan, Chan Lek; Nguyen, Thuc Nghi; Beier, Kevin T.; Hörmann, Nikolai; Chang, Wei-Cheng; Zhang, Zhe (2017). "Identification of preoptic sleep neurons using retrograde labelling and gene profiling". Nature. 545 (7655): 477–481. Bibcode:2017Natur.545..477C. doi:10.1038/nature22350. PMC   5554302 . PMID   28514446.
  13. 1 2 Mohammad, Farhan; Stewart, James C; Ott, Stanislav; Chlebikova, Katarina; Chua, Jia Yi; Koh, Tong-Wey; Ho, Joses; Claridge-Chang, Adam (2017). "Optogenetic inhibition of behavior with anion channelrhodopsins". Nature Methods. 14 (3): 271–274. doi:10.1038/nmeth.4148. PMID   28114289. S2CID   4133602.
  14. 1 2 Mauss, Alex S.; Busch, Christian; Borst, Alexander (2017-10-23). "Optogenetic Neuronal Silencing in Drosophila during Visual Processing". Scientific Reports. 7 (1): 13823. Bibcode:2017NatSR...713823M. doi:10.1038/s41598-017-14076-7. ISSN   2045-2322. PMC   5653863 . PMID   29061981.
  15. ↑ Govorunova, Elena G.; Cunha, Shane R.; Sineshchekov, Oleg A.; Spudich, John L. (2016-09-15). "Anion channelrhodopsins for inhibitory cardiac optogenetics". Scientific Reports. 6 (1) 33530. Bibcode:2016NatSR...633530G. doi:10.1038/srep33530. ISSN   2045-2322. PMC   5024162 . PMID   27628215.
  16. 1 2 Mardinly, Alan R.; Oldenburg, Ian Antón; Pégard, Nicolas C.; Sridharan, Savitha; Lyall, Evan H.; Chesnov, Kirill; Brohawn, Stephen G.; Waller, Laura; Adesnik, Hillel (2018-04-30). "Precise multimodal optical control of neural ensemble activity". Nature Neuroscience. 21 (6): 881–893. doi:10.1038/s41593-018-0139-8. ISSN   1097-6256. PMC   5970968 . PMID   29713079.
  17. 1 2 Mahn, Mathias; Gibor, Lihi; Malina, Katayun Cohen-Kashi; Patil, Pritish; Printz, Yoav; Oring, Shir; Levy, Rivka; Lampl, Ilan; Yizhar, Ofer (2018). "High-efficiency optogenetic silencing with soma-targeted anion-conducting channelrhodopsins". Nature Communications. 9 (1): 4125. Bibcode:2018NatCo...9.4125M. doi: 10.1038/s41467-018-06511-8 . PMC   6175909 . PMID   30297821.
  18. ↑ Wietek, Jonas; Broser, Matthias; Krause, Benjamin S.; Hegemann, Peter (2016-02-19). "Identification of a Natural Green Light Absorbing Chloride Conducting Channelrhodopsin from Proteomonas sulcata". Journal of Biological Chemistry. 291 (8): 4121–4127. doi: 10.1074/jbc.M115.699637 . ISSN   0021-9258. PMC   4759187 . PMID   26740624.
  19. ↑ Govorunova, Elena G.; Sineschekov, Oleg A.; Spudich, John L. (2016-02-01). "Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin". Photochemistry and Photobiology. 92 (2): 257–263. doi:10.1111/php.12558. PMC   4914479 . PMID   26686819.
  20. 1 2 Wietek, Jonas; Rodriguez-Rozada, Silvia; Tutas, Janine; Tenedini, Federico; Grimm, Christiane; Oertner, Thomas G.; Soba, Peter; Hegemann, Peter; Wiegert, J. Simon (Nov 2017). "Anion-conducting channelrhodopsins with tuned spectra and modified kinetics engineered for optogenetic manipulation of behavior". Scientific Reports. 7 (1): 14957. Bibcode:2017NatSR...714957W. doi:10.1038/s41598-017-14330-y. ISSN   2045-2322. PMC   5668261 . PMID   29097684.
  21. ↑ Oppermann, Johannes; Fischer, Paul; Silapetere, Arita; Liepe, Bernhard; Rodriguez-Rozada, Silvia; Flores-Uribe, José; Peter, Enrico; Keidel, Anke; Vierock, Johannes; Kaufmann, Joel; Broser, Matthias (2019-07-25). "MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins". Nature Communications. 10 (1): 3315. Bibcode:2019NatCo..10.3315O. doi:10.1038/s41467-019-11322-6. ISSN   2041-1723. PMC   6658528 . PMID   31346176.
  22. ↑ Mahn, Mathias; Prigge, Matthias; Ron, Shiri; Levy, Rivka; Yizhar, Ofer (2016). "Biophysical constraints of optogenetic inhibition at presynaptic terminals". Nature Neuroscience. 19 (4): 554–556. doi:10.1038/nn.4266. PMC   4926958 . PMID   26950004.
  23. ↑ Vierock, Johannes; Rodriguez-Rozada, Silvia; Dieter, Alexander; Pieper, Florian; Sims, Ruth; Tenedini, Federico; Bergs, Amelie C. F.; Bendifallah, Imane; Zhou, Fangmin; Zeitzschel, Nadja; Ahlbeck, Joachim (2021-07-26). "BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons". Nature Communications. 12 (1): 4527. Bibcode:2021NatCo..12.4527V. doi:10.1038/s41467-021-24759-5. ISSN   2041-1723. PMC   8313717 . PMID   34312384.
  24. ↑ Wiegert, J. Simon; Mahn, Mathias; Prigge, Matthias; Printz, Yoav; Yizhar, Ofer (2017). "Silencing Neurons: Tools, Applications, and Experimental Constraints". Neuron. 95 (3): 504–529. doi:10.1016/j.neuron.2017.06.050. PMC   5830081 . PMID   28772120.
  25. ↑ Evanko, Daniel (2014). "Neuroscience: A better way to turn off neurons". Nature Methods. 11 (6): 608. doi: 10.1038/nmeth.2988 . S2CID   1699434.
  26. ↑ Berndt, Andre; Deisseroth, Karl (2015-08-07). "Expanding the optogenetics toolkit". Science. 349 (6248): 590–591. Bibcode:2015Sci...349..590B. doi:10.1126/science.aac7889. ISSN   0036-8075. PMC   4776750 . PMID   26250674.
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