Limits...
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

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
Cartoon of the Channelrhodopsin 7TM-fragmentThe structure is drawn according to the data of Kato et al (2012) with key residues shown in color: voltage sensor E123 (cyan), residues of the access channel (magenta), central gate (blue), and inner gate (orange), OH-cluster green, and the retinal Schiff base is seen in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3569634&req=5

fig02: Cartoon of the Channelrhodopsin 7TM-fragmentThe structure is drawn according to the data of Kato et al (2012) with key residues shown in color: voltage sensor E123 (cyan), residues of the access channel (magenta), central gate (blue), and inner gate (orange), OH-cluster green, and the retinal Schiff base is seen in red.

Mentions: 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. The light-absorbing chromophore retinal, a vitamin A derivative, is embedded within the hydrophobic center of the seven helices (Fig 2). The retinal is connected to a conserved lysine via a Schiff base linkage (C=N), which is protonated to shift the absorption into the visible range of the spectrum. The colour of the protonated retinal Schiff base (RSBH+) is fine-tuned by the distance of the negatively charged counter ion that together form the active site (Fig 2) and the location of a few polar residues around the retinal polyene chain. Light absorption by retinal leads to isomerization, followed by a protein conformational change and opening of the ion pore (Fig 1D). In the light-activated ion pumps, bacteriorhodopsin and halorhodopsin undergo similar conformational changes, which lead to active proton export and Cl− import, respectively. Interestingly, internal and external pHs strongly influence ChR2 channel closing and recovery from desensitization (Nagel et al, 2003).


From channelrhodopsins to optogenetics.

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

Cartoon of the Channelrhodopsin 7TM-fragmentThe structure is drawn according to the data of Kato et al (2012) with key residues shown in color: voltage sensor E123 (cyan), residues of the access channel (magenta), central gate (blue), and inner gate (orange), OH-cluster green, and the retinal Schiff base is seen in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Cartoon of the Channelrhodopsin 7TM-fragmentThe structure is drawn according to the data of Kato et al (2012) with key residues shown in color: voltage sensor E123 (cyan), residues of the access channel (magenta), central gate (blue), and inner gate (orange), OH-cluster green, and the retinal Schiff base is seen in red.
Mentions: 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. The light-absorbing chromophore retinal, a vitamin A derivative, is embedded within the hydrophobic center of the seven helices (Fig 2). The retinal is connected to a conserved lysine via a Schiff base linkage (C=N), which is protonated to shift the absorption into the visible range of the spectrum. The colour of the protonated retinal Schiff base (RSBH+) is fine-tuned by the distance of the negatively charged counter ion that together form the active site (Fig 2) and the location of a few polar residues around the retinal polyene chain. Light absorption by retinal leads to isomerization, followed by a protein conformational change and opening of the ion pore (Fig 1D). In the light-activated ion pumps, bacteriorhodopsin and halorhodopsin undergo similar conformational changes, which lead to active proton export and Cl− import, respectively. Interestingly, internal and external pHs strongly influence ChR2 channel closing and recovery from desensitization (Nagel et al, 2003).

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