ChR2

Target Synonyms and Classification: Channelrhodopsin 2 (ChR2) belongs to the Ion-translocating Microbial Rhodopsin Family (MRF, TCDB: 3.E.1), which catalyzes light-driven ion translocation across microbial cytoplasmic membranes or serve as light receptors.

Function and Mechanism: ChR2 is a light-gated cation-selective ion channel that transports both monovalent and divalent cations. Upon light adsorption (λmax = 480 nm), the retinal chromophore undergoes a conformational change which is linked to pore opening. After milliseconds, the retinal relaxes, closing the pore and stopping the flow of ions.

Organism and Localization: Channelrhodopsins are found in unicellular green algae. ChR2 from the model organism Chlamydomonas reinhardtii was among the first discovered channelrhodopsins. ChR2 shows dynamic localiation and shuttles between flagella and eyespot of Chlamydomonas in a light-dependent manner: After prolonged light exposure, ChR2 is only found within the eyespot and mediates the photoreceptor current. In the dark, ChR2 additionally triggers the flagellar photocurrent that brings the change in calcium flux to control the movement.

Substrates and Inhibitors: ChR2 is cation-selective and transports both, monovalent and divalent cations (H+, Na+, K+, Ca2+). It is rather nonspecific - especially at the beginning of a light pulse. But it was shown that ChR2 becomes more specific for protons during longer periods of light exposure, reflecting a second opening state.

Related Transporters: All Rhodopsins share the properties of a covalently bound retinal coupling light adsorption with ion translocation. But in opposite to the light gated ion channel ChR2, most Rhodopsins function as light driven ion pumps, e.g. Bacteriorhodopsin. ChR1 from Chlamydomonas reinhardtii differs from ChR2: it is proton-selective and shows smaller photocurrents. Channelrhodopsins are often used in optogenitics: They enable light to control electrical excitability of membranes. Different genetic modifications – point mutations and Chimeras between different Channelrhodopopsin variants - are known that alter the properties of ChR2: most importantly absorption spectra (ReaChR and Chrimson show red spectral shifts), ion selectivity (CatCh prefers Ca2+; ChloC translocates Cl-), translocation speed (ChETA has faster kinetics) and current amplitude (ChIEF demonstrates largest photocurrents).

Data and Applications

ChR2 - Variation of light intensity

ChR2   Variation of light intensity Figure2Icon N1   SURFE2R N1 data and applications:

We observed saturation kinetics for the light-induced ChR2 activity with increasing LED intensities using a prototypical SURFE2R N1 add-on for activation by light.

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ChR2 - Symmetry of transport

ChR2   Symmetry of transport Figure3Icon N1   SURFE2R N1 data and applications:

Depending on the direction of the Na+ gradient, we observed positive or negative transient currents after ChR2 activation by light. A negative control without ChR2 shows no current signal upon light activation.

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ChR2 - Stability of Retinal binding

ChR2 Stability of Retinal binding Figure1Icon N1   SURFE2R N1 data and applications:

We investigated the effects of retinal addition and removal on light-induced ChR2 activity using a prototypical SURFE2R N1 add-on for activation by light.

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ChR2 - Membrane potential as driving force

ChR2   Membrane potential as driving force Figure4Icon N1   SURFE2R N1 data and applications:

We investigated the effect of a membrane potential - generated by Na+/Ca2+ exchange activity of NCX1 - on the signal amplitude of light induced Na+ flux by ChR2.

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ChR2 - Exposure time and frequency of light

ChR2   Exposure time and frequency of light Figure5Icon N1   SURFE2R N1 data and applications:

We investigated the influence of the duration and frequency of the light pulses for ChR2 activation on the stability of the ChR2 signals over time.

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ChR2 - Cell-based assay optimization

ChR2   Cell based assay optimization Figure6Icon N1   SURFE2R N1 data and applications:

For assay optimization we checked the influence on cell suspension volume we have attached to one single sensor on the signal-to-noise ratio during the SURFE2R experiment.

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Cardiomyocytes - Optogenetics meets cardiac safety

Icon CE   CardioExcyte Optogenetics 1CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

The stimulating optical lid, CardioExcyte 96 SOL, uses LEDs for spatially uniform stimulation of cells transfected with light-gated ion channels such as Channelrhodopsin2 (ChR2).

Right graph: LPM – Light pulse per minute plotted against the recorded beat rate (average of 96 wells). ChR2 transfected Cor.4U cells are following the optical pace rate.

Cardiomyocytes - Myocyte phase II study: CiPA conform analysis and arrhythmia detection

Icon CE   CardioExcyte CiPAII 1CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

Nanion developed a CiPA conform analysis for the Myocyte phase II study. The feature comes along is included in our CiPA analysis routine. Automated arrhythmia detection is just one highlight out of many when it comes to the CardioExcyte 96 software.

Cardiomyocytes - Channelrhodopsin 2 (ChR2) transfected Cor.4U cells and optical pacing

Icon CE   CardioExcyte Optogenetics 2CardioExcyte data and applications:
Cells were kindly provided by Axiogenesis.

ChR2 transfected Cor.4U cells are following the optical pace rate.

Raw data traces upon a 1 Hz, 1.5 Hz, 2 Hz., 2.5 Hz and 3 Hz stimulation rate, extracellular field potentials (top) and impedance (bottom).

 

 

 

Publications

2020 - Wireless Charging Electrochemiluminescence System for Ionic Channel Manipulation in Living Cells

icon pap   Port-a-Patch publication in ACS Applied Materials & Interfaces (2020)

Authors:
Peng K., Liu S., Lv F., Fu X., Hussain S., Zhao H., Liu L., Wang S.

2018 - An update on the advancing high-throughput screening techniques for patch clamp-based ion channel screens: implications for drug discovery

icon sp96   SyncroPatch 384/768PE (a predecessor model of SyncroPatch 384/768i) and   icon pl   Patchliner publication in Expert Opinion on Drug Discovery

Authors: 
Obergrussberger A., Goetze T.A., Brinkwirth N., Becker N., Friis S., Rapedius M., Haarmann C., Rinke-Weiß I., Stölzle-Feix S., Brüggemann A., George M., Fertig N.

2017 - Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation

Icon CE  CardioExcyte 96 publication in International Journal of Molecular Sciences

Authors:
Rehnelt S., Malan D., Juhasz K., Wolters B., Doerr L., Beckler M., Kettenhofen R., Bohlen H., Bruegmann T., Sasse P.

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