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Channelrhodopsin-2 (ChR2) is a cation-selective light-gated channel from Chlamydomonas reinhardtii (Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 2003;100:13940-5), which has become a powerful tool in optogenetics. Two-dimensional crystals of the slow photocycling C128T ChR2 mutant were exposed to 473 nm light and rapidly frozen to trap the open state. Projection difference maps at 6Å resolution show the location, extent and direction of light-induced conformational changes in ChR2 during the transition from the closed state to the ion-conducting open state. Difference peaks indicate that transmembrane helices (TMHs) TMH2, TMH6 and TMH7 reorient or rearrange during the photocycle. No major differences were found near TMH3 and TMH4 at the dimer interface. While conformational changes in TMH6 and TMH7 are known from other microbial-type rhodopsins, our results indicate that TMH2 has a key role in light-induced channel opening and closing in ChR2.
The desensitized channelrhodopsin-2 photointermediate contains 13 -cis, 15 -syn retinal Schiff base
(2021)
Channelrhodopsin-2 (ChR2) is a light-gated cation channel and was used to lay the foundations of optogenetics. Its dark state X-ray structure has been determined in 2017 for the wild-type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP-enhanced solid-state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle.
Optogenetic stimulation of inhibitory interneurons has become a commonly used strategy for silencing neuronal activity. This is typically achieved using transgenic mice expressing excitatory opsins in inhibitory interneurons throughout the brain, raising the question of how spatially extensive the resulting inhibition is. Here, we characterize neuronal silencing in VGAT-ChR2 mice, which express channelrhodopsin-2 in inhibitory interneurons, as a function of light intensity and distance from the light source in several cortical and subcortical regions. We show that light stimulation, even at relatively low intensities, causes inhibition not only in brain regions targeted for silencing but also in their subjacent areas. In contrast, virus-mediated expression of an inhibitory opsin enables robust silencing that is restricted to the region of opsin expression. Our results reveal important constraints on using inhibitory interneuron activation to silence neuronal activity and emphasize the necessity of carefully controlling light stimulation parameters when using this silencing strategy.