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iChloC structure
Figure 1: It took 5 point mutations to create iChloC from cation-conducting Channelrhodopsin-2. [1]

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.

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. [9]

Variants

name species of origin absorption reference properties, applications
slowChloC Chlamydomonas reinhardtii blue Wietek et al. 2014 [3] first generation, mixed conductance
iC1C2 Chlamydomonas reinhardtii blue Berndt et al. 2014 [10] first generation, mixed conductance
iChloC Chlamydomonas reinhardtii blue Wietek et al. 2015 [1] inhibition of perception in mice [2]
iC++ Chlamydomonas reinhardtii blue Berndt et al. 2016 [11] inhibition of sleep in mice [12]
GtACR1 Guillardia theta green Govorunova 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 on Kato et al. 2018 [7] very fast closing, large currents

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

GtACR2 Guillardia theta blue Govorunova et al. 2015 [4] inhibition of behavior in Drosophila [13] inhibition of fear extinction in mice [17]
PsACR1 Proteomonas sulcata green Wietek et al. 2016, [18] Govorunova et al. 2016 [19] large currents
ZipACR Proteomonas sulcata green Govorunova et al. 2017 [5] very fast
RapACR Rhodomonas salina green Govorunova 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-red Wietek et al. 2017 [20] stop locomotion of Drosophila larvae
MerMAIDs unknown green Oppermann 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. ^ a b c 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. doi: 10.1038/srep14807. ISSN  2045-2322. PMC  4595828. PMID  26443033.
  2. ^ a b c Takahashi, Naoya; Oertner, Thomas G.; Hegemann, Peter; Larkum, Matthew E. (2016-12-23). "Active cortical dendrites modulate perception". Science. 354 (6319): 1587–1590. doi: 10.1126/science.aah6066. ISSN  0036-8075. PMID  28008068. S2CID  28317052.
  3. ^ a b 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. doi: 10.1126/science.1249375. ISSN  0036-8075. PMID  24674867. S2CID  206554245.
  4. ^ a b c 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. doi: 10.1126/science.aaa7484. ISSN  0036-8075. PMC  4764398. PMID  26113638.
  5. ^ a b 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. doi: 10.1038/srep43358. ISSN  2045-2322. PMC  5335703. PMID  28256618.
  6. ^ a b c 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.
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  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. 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.
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  11. ^ a b 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. doi: 10.1073/pnas.1523341113. ISSN  0027-8424. PMC  4743797. PMID  26699459.
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  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. doi: 10.1038/srep33530. ISSN  2045-2322. PMC  5024162. PMID  27628215.
  16. ^ a b 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. ^ a b 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. 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.
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