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

Related Research Articles

Channelrhodopsins are a subfamily of retinylidene proteins (rhodopsins) that function as light-gated ion channels. They serve as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light. Expressed in cells of other organisms, they enable light to control electrical excitability, intracellular acidity, calcium influx, and other cellular processes. Channelrhodopsin-1 (ChR1) and Channelrhodopsin-2 (ChR2) from the model organism Chlamydomonas reinhardtii are the first discovered channelrhodopsins. Variants that are sensitive to different colors of light or selective for specific ions have been cloned from other species of algae and protists.

<span class="mw-page-title-main">Photostimulation</span>

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<span class="mw-page-title-main">Halorhodopsin</span> Family of transmembrane proteins


Halorhodopsin is a seven-transmembrane retinylidene protein from microbial rhodopsin family. It is a chloride-specific light-gated ion pump found in archaea known as halobacteria. It is activated by green light wavelengths of approximately 578nm. Halorhodopsin also shares sequence similarity to channelrhodopsin, another light-driven ion channel.

Retinylidene proteins, or rhodopsins in a broad sense, are proteins that use retinal as a chromophore for light reception. They are the molecular basis for a variety of light-sensing systems from phototaxis in flagellates to eyesight in animals. Retinylidene proteins include all forms of opsin and rhodopsin. While rhodopsin in the narrow sense refers to a dim-light visual pigment found in vertebrates, usually on rod cells, rhodopsin in the broad sense refers to any molecule consisting of an opsin and a retinal chromophore in the ground state. When activated by light, the chromophore is isomerized, at which point the molecule as a whole is no longer rhodopsin, but a related molecule such as metarhodopsin. However, it remains a retinylidene protein. The chromophore then separates from the opsin, at which point the bare opsin is a retinylidene protein. Thus, the molecule remains a retinylidene protein throughout the phototransduction cycle.

Light-gated ion channels are a family of ion channels regulated by electromagnetic radiation. Other gating mechanisms for ion channels include voltage-gated ion channels, ligand-gated ion channels, mechanosensitive ion channels, and temperature-gated ion channels. Most light-gated ion channels have been synthesized in the laboratory for study, although two naturally occurring examples, channelrhodopsin and anion-conducting channelrhodopsin, are currently known. Photoreceptor proteins, which act in a similar manner to light-gated ion channels, are generally classified instead as G protein-coupled receptors.

Optogenetics is a biological technique to control the activity of neurons or other cell types with light. This is achieved by expression of light-sensitive ion channels, pumps or enzymes specifically in the target cells. On the level of individual cells, light-activated enzymes and transcription factors allow precise control of biochemical signaling pathways. In systems neuroscience, the ability to control the activity of a genetically defined set of neurons has been used to understand their contribution to decision making, learning, fear memory, mating, addiction, feeding, and locomotion. In a first medical application of optogenetic technology, vision was partially restored in a blind patient.

<span class="mw-page-title-main">Axon terminal</span>

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<span class="mw-page-title-main">Karl Deisseroth</span> American optogeneticist (born 1971)

Karl Alexander Deisseroth is an American scientist. He is the D.H. Chen Professor of Bioengineering and of psychiatry and behavioral sciences at Stanford University.

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<span class="mw-page-title-main">Peter Hegemann</span> German biophysicist

Peter Hegemann is a Hertie Senior Research Chair for Neurosciences and a Professor of Experimental Biophysics at the Department of Biology, Faculty of Life Sciences, Humboldt University of Berlin, Germany. He is known for his discovery of channelrhodopsin, a type of ion channels regulated by light, thereby serving as a light sensor. This created the field of optogenetics, a technique that controls the activities of specific neurons by applying light. He has received numerous accolades, including the Rumford Prize, the Shaw Prize in Life Science and Medicine, and the Albert Lasker Award for Basic Medical Research.

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<span class="mw-page-title-main">Microbial rhodopsin</span> Retinal-binding proteins

Microbial rhodopsins, also known as bacterial rhodopsins, are retinal-binding proteins that provide light-dependent ion transport and sensory functions in halophilic and other bacteria. They are integral membrane proteins with seven transmembrane helices, the last of which contains the attachment point for retinal.

<span class="mw-page-title-main">Georg Nagel</span>

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<span class="mw-page-title-main">Photoactivated adenylyl cyclase</span>

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Lisa Gunaydin is an American neuroscientist and assistant professor at the Weill Institute for Neurosciences at the University of California San Francisco. Gunaydin helped discover optogenetics in the lab of Karl Deisseroth and now uses this technique in combination with neural and behavioral recordings to probe the neural circuits underlying emotional behaviors.

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Optogenetics began with methods to alter neuronal activity with light, using e.g. channelrhodopsins. In a broader sense, optogenetic approaches also include the use of genetically encoded biosensors to monitor the activity of neurons or other cell types by measuring fluorescence or bioluminescence. Genetically encoded calcium indicators (GECIs) are used frequently to monitor neuronal activity, but other cellular parameters such as membrane voltage or second messenger activity can also be recorded optically. The use of optogenetic sensors is not restricted to neuroscience, but plays increasingly important roles in immunology, cardiology and cancer research.

References

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