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From channelrhodopsins to optogenetics.

Hegemann P, Nagel G - EMBO Mol Med (2013)

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Experimental Biophysics, Humboldt-University of Berlin, Berlin, Germany. hegemann@rz.hu-berlin.de

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We did not expect that research on the molecular mechanism of algal phototaxis or archaeal light-driven ion transport might interest readers of a medical journal when we conceived and performed our experiments a decade ago... On the other hand, it did not escape our attention that channelrhodopsin is helping an ever-increasing number of researchers to address their specific questions... The discovery of channelrhodopsin is based on two quite different research fields, studies on living algae and experiments on reconstituted microbial rhodopsins... Ken substantiated his claim by restoring behavioural light responses in blind algae by complementation with retinal and retinal analogues (Foster et al, )... However, the photoreceptor field did not really understand the importance of the claim and progress remained slow... We named these new genes channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2; Nagel et al,, ; Fig 1C and D)... The success of ChR2 encouraged us and a number of neurobiologists to test halorhodopsin, a light-driven chloride importer and membrane hyperpolarizer, as an additional optogenetic tool for action potential suppression, which worked astonishingly well (Zhang et al, ). »…demonstrated the functionality of ChR2 in the retina of blind mice, hippocampal neurons, spine of living chicken embryos, PC12 cells, mouse brain slices and transgenic worms…« ChRs are composed of seven trans-membrane helices that form the ion channel and a long C-terminal extension of unknown function, which is routinely omitted for optogenetic purposes... We now know that this reaction path differs from the opening path and that the kinetics of dark state recovery is many orders of magnitudes slower... Besides the OH-cluster, two residues, C128 and D156 (DC-pair in Fig 2) are of fundamental importance for both channel opening and closing, and mutation of either residue results in a dramatic increase of the open state(s)' lifetime... We may be able to widen the pore by molecular engineering, but presumably at the cost of destabilization and thermal activation in darkness... Selectivity can be changed towards higher or exclusive H conductance as found naturally in ChR from the halotolerant alga Dunaliella salina (Zhang et al, )... Better solutions for targeting ChRs into membrane subareas will be found, directing them into organelles, making them bimodal switchable, controlling expression more accurately, and guaranteeing better turnover and photostability for retinal prosthesis and vision in bright light.

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The discovery of channelrhodopsinsPhototaxis of the Chlamydomonas wild type strain CW2 and the channelrhodopsin-defective mutant H17.Electrophysiological recording from oocytes (in the centre, with two electrodes, left and right) allows investigation of light-induced (via light guide, below oocyte) currents, e.g. mediated by rhodopsins.Light-induced currents mediated by channelrhodopsin-2 (two illuminations for 1 s, blue bars) at −100 mV.Model of channelrhodopsin opening, following light absorption and isomerization of covalently bound retinal.
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fig01: The discovery of channelrhodopsinsPhototaxis of the Chlamydomonas wild type strain CW2 and the channelrhodopsin-defective mutant H17.Electrophysiological recording from oocytes (in the centre, with two electrodes, left and right) allows investigation of light-induced (via light guide, below oocyte) currents, e.g. mediated by rhodopsins.Light-induced currents mediated by channelrhodopsin-2 (two illuminations for 1 s, blue bars) at −100 mV.Model of channelrhodopsin opening, following light absorption and isomerization of covalently bound retinal.

Mentions: A number of researchers have characterized the swimming behaviour and light responses of motile microalgae over at least 140 years (Fig 1A). Early studies on green microalgae root back to L.G. Treviranus (Treviranus, 1817) and behavioural responses were described by A. Famintzin from St. Petersburg University in 1878 (Famintzin, 1878). During helical swimming of the green alga Chlamydomonas, its orange eye signals to the flagella to alter the flagellar beating plane (Mast, 1916). Researchers at Stanford University implicated Mg2+ and Ca2+ in the behavioural responses and identified the role of Ca2+ influx in flagellar beat frequency changes (Halldal, 1957, Schmidt & Eckert, 1976). Then Oleg Sineshchekov from Moscow State University recorded electrical light responses from Haematococcus pluvialis, an alga known for the production of the antioxidant Astaxanthine (Litvin et al, 1978). Oleg used a suction pipette technique applied at the time by Dennis Baylor for recording photocurrents from bovine photoreceptor rods and cones. But Oleg's publication gave no hints about the type of photoreceptor involved. Kenneth W. Foster however, a physicist at Mount Sinai School of Medicine re-analysed published action spectra for phototactic movement of algae and postulated that the sensory photoreceptor is rhodopsin (Foster & Smyth, 1980). Ken substantiated his claim by restoring behavioural light responses in blind algae by complementation with retinal and retinal analogues (Foster et al, 1984). However, the photoreceptor field did not really understand the importance of the claim and progress remained slow. Years later, Peter Hegemann's former graduate student Hartmann Harz recorded photocurrents from Chlamydomonas by revitalizing Oleg's suction pipette technique for a Chlamydomonas cell wall-deficient mutant. He recorded action spectra, which led to the proposal that the photocurrents were mediated by a rhodopsin, the photoreceptor that also mediates phototaxis and phobic responses (Harz & Hegemann, 1991). The ultra-fast appearance of the photoreceptor current suggested that the photoreceptor and ion channel were intimately linked, forming a single protein complex (Braun & Hegemann, 1999).


From channelrhodopsins to optogenetics.

Hegemann P, Nagel G - EMBO Mol Med (2013)

The discovery of channelrhodopsinsPhototaxis of the Chlamydomonas wild type strain CW2 and the channelrhodopsin-defective mutant H17.Electrophysiological recording from oocytes (in the centre, with two electrodes, left and right) allows investigation of light-induced (via light guide, below oocyte) currents, e.g. mediated by rhodopsins.Light-induced currents mediated by channelrhodopsin-2 (two illuminations for 1 s, blue bars) at −100 mV.Model of channelrhodopsin opening, following light absorption and isomerization of covalently bound retinal.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569634&req=5

fig01: The discovery of channelrhodopsinsPhototaxis of the Chlamydomonas wild type strain CW2 and the channelrhodopsin-defective mutant H17.Electrophysiological recording from oocytes (in the centre, with two electrodes, left and right) allows investigation of light-induced (via light guide, below oocyte) currents, e.g. mediated by rhodopsins.Light-induced currents mediated by channelrhodopsin-2 (two illuminations for 1 s, blue bars) at −100 mV.Model of channelrhodopsin opening, following light absorption and isomerization of covalently bound retinal.
Mentions: A number of researchers have characterized the swimming behaviour and light responses of motile microalgae over at least 140 years (Fig 1A). Early studies on green microalgae root back to L.G. Treviranus (Treviranus, 1817) and behavioural responses were described by A. Famintzin from St. Petersburg University in 1878 (Famintzin, 1878). During helical swimming of the green alga Chlamydomonas, its orange eye signals to the flagella to alter the flagellar beating plane (Mast, 1916). Researchers at Stanford University implicated Mg2+ and Ca2+ in the behavioural responses and identified the role of Ca2+ influx in flagellar beat frequency changes (Halldal, 1957, Schmidt & Eckert, 1976). Then Oleg Sineshchekov from Moscow State University recorded electrical light responses from Haematococcus pluvialis, an alga known for the production of the antioxidant Astaxanthine (Litvin et al, 1978). Oleg used a suction pipette technique applied at the time by Dennis Baylor for recording photocurrents from bovine photoreceptor rods and cones. But Oleg's publication gave no hints about the type of photoreceptor involved. Kenneth W. Foster however, a physicist at Mount Sinai School of Medicine re-analysed published action spectra for phototactic movement of algae and postulated that the sensory photoreceptor is rhodopsin (Foster & Smyth, 1980). Ken substantiated his claim by restoring behavioural light responses in blind algae by complementation with retinal and retinal analogues (Foster et al, 1984). However, the photoreceptor field did not really understand the importance of the claim and progress remained slow. Years later, Peter Hegemann's former graduate student Hartmann Harz recorded photocurrents from Chlamydomonas by revitalizing Oleg's suction pipette technique for a Chlamydomonas cell wall-deficient mutant. He recorded action spectra, which led to the proposal that the photocurrents were mediated by a rhodopsin, the photoreceptor that also mediates phototaxis and phobic responses (Harz & Hegemann, 1991). The ultra-fast appearance of the photoreceptor current suggested that the photoreceptor and ion channel were intimately linked, forming a single protein complex (Braun & Hegemann, 1999).

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Experimental Biophysics, Humboldt-University of Berlin, Berlin, Germany. hegemann@rz.hu-berlin.de

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

We did not expect that research on the molecular mechanism of algal phototaxis or archaeal light-driven ion transport might interest readers of a medical journal when we conceived and performed our experiments a decade ago... On the other hand, it did not escape our attention that channelrhodopsin is helping an ever-increasing number of researchers to address their specific questions... The discovery of channelrhodopsin is based on two quite different research fields, studies on living algae and experiments on reconstituted microbial rhodopsins... Ken substantiated his claim by restoring behavioural light responses in blind algae by complementation with retinal and retinal analogues (Foster et al, )... However, the photoreceptor field did not really understand the importance of the claim and progress remained slow... We named these new genes channelrhodopsin-1 (ChR1) and channelrhodopsin-2 (ChR2; Nagel et al,, ; Fig 1C and D)... The success of ChR2 encouraged us and a number of neurobiologists to test halorhodopsin, a light-driven chloride importer and membrane hyperpolarizer, as an additional optogenetic tool for action potential suppression, which worked astonishingly well (Zhang et al, ). »…demonstrated the functionality of ChR2 in the retina of blind mice, hippocampal neurons, spine of living chicken embryos, PC12 cells, mouse brain slices and transgenic worms…« ChRs are composed of seven trans-membrane helices that form the ion channel and a long C-terminal extension of unknown function, which is routinely omitted for optogenetic purposes... We now know that this reaction path differs from the opening path and that the kinetics of dark state recovery is many orders of magnitudes slower... Besides the OH-cluster, two residues, C128 and D156 (DC-pair in Fig 2) are of fundamental importance for both channel opening and closing, and mutation of either residue results in a dramatic increase of the open state(s)' lifetime... We may be able to widen the pore by molecular engineering, but presumably at the cost of destabilization and thermal activation in darkness... Selectivity can be changed towards higher or exclusive H conductance as found naturally in ChR from the halotolerant alga Dunaliella salina (Zhang et al, )... Better solutions for targeting ChRs into membrane subareas will be found, directing them into organelles, making them bimodal switchable, controlling expression more accurately, and guaranteeing better turnover and photostability for retinal prosthesis and vision in bright light.

Show MeSH
Related in: MedlinePlus