Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs

Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely related to ion pump rhodopsins than other channelrhodopsins. ChRmine displays unique properties favorable for optogenetics including high light sensitivity, a red-shifted activation spectrum, cation selectivity, and large photocurrents while its slow closing kinetics impede some applications. The structural basis for ChRmine function, or that of any other BCCR, is unknown. Here, we present cryo-EM structures of ChRmine in lipid nanodiscs in apo (opsin) and retinal-bound (rhodopsin) forms. The structures reveal an unprecedented trimeric architecture with a lipid filled central pore. Large electronegative cavities on either side of the membrane facilitate high conductance and selectivity for cations over protons. The retinal binding pocket structure suggests spectral and kinetic properties could be tuned with mutations and we identify ChRmine variants with two-fold increased and ten-fold decreased closing rates. These results provide insight into structural features that generate an ultra-potent microbial opsin and provide a platform for rational engineering of channelrhodopsins with improved properties that could expand the scale, depth, and precision of optogenetic manipulations.

RubyACRs, nonalgal anion channelrhodopsins with highly red-shifted absorption

Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulea and the other from haptophyte algae. Four closely related labyrinthulea ACRs, named RubyACRs here, exhibit a unique retinal-binding pocket that creates spectral sensitivities with maxima at 590 to 610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins thus far reported. We identified three spectral tuning residues critical for the red-shifted absorption. Photocurrents recorded from the RubyACR from Aurantiochytrium limacinum (designated AlACR1) under single-turnover excitation exhibited biphasic decay, the rate of which was only weakly voltage dependent, in contrast to that in previously characterized cryptophyte ACRs, indicating differences in channel gating mechanisms between the two ACR families. Moreover, in A. limacinum we identified three ACRs with absorption maxima at 485, 545, and 590 nm, indicating color-sensitive photosensing with blue, green, and red spectral variation of ACRs within individual species of the labyrinthulea family. We also report functional energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs.

Unique retinal binding pocket of primate blue-sensitive visual pigment

ABSTRACTThe visual pigments of humans contain 11-cis retinal as the chromophore of light perception, and its photoisomerization to the all-trans form initiates visual excitation in our eyes. It is well known that three isomeric states of retinal (11-cis, all-trans, and 9-cis) are in photoequilibrium at very low temperatures such as 77 K. Here we report the lack of formation of the 9-cis form in monkey blue (MB) at 77 K, as revealed by light-induced difference FTIR spectroscopy. This indicates that the chromophore binding pocket of MB does not accommodate the 9-cis form, even though it accommodates the all-trans form by twisting the chromophore. Mutation of the blue-specific tyrosine at position 265 into tryptophan, which is highly conserved in other animal rhodopsins, led to formation of the 9-cis form in MB, suggesting that Y265 is one of the determinants of the unique photochemistry in blue pigments. We also found that 9-cis retinal does not bind to MB opsin, implying that the chromophore binding pocket does not accommodate the 9-cis form at physiological temperature. The unique property of MB is discussed based on the present results.

RubyACRs, non-algal anion channelrhodopsins with highly red-shifted absorption

Channelrhodopsins are light-gated ion channels widely used to control neuronal firing with light (optogenetics). We report two previously unknown families of anion channelrhodopsins (ACRs), one from the heterotrophic protists labyrinthulomycetes and the other from haptophyte algae. Four closely related labyrinthulomycete ACRs, named RubyACRs here, exhibit a unique retinal binding pocket that creates spectral sensitivities with maxima at 590-610 nm, the most red-shifted channelrhodopsins known, long-sought for optogenetics, and more broadly the most red-shifted microbial rhodopsins so far reported. We identified three spectral tuning residues critical for the red-shifted absorption. Photocurrents recorded from the RubyACR from Aurantiochytrium limacinum (designated AlACR1) under single-turnover excitation exhibited biphasic decay, the rate of which was only weakly voltage-dependent, in contrast to that in previously characterized cryptophyte ACRs, indicating differences in channel gating mechanisms between the two ACR families. Moreover, in A. limacinum we identified three ACRs with absorption maxima at 485, 545, and 590 nm, indicating color-sensitive photosensing with blue, green and red spectral variation of ACRs within individual species of the labyrinthulomycete family. We also report energy transfer from a cytoplasmic fluorescent protein domain to the retinal chromophore bound within RubyACRs, not seen in similar constructs in other channelrhodopsins.Significance StatementOur identification and characterization of two ACR families, one from non-photosynthetic microorganisms, shows that light-gated anion conductance is more widely spread among eukaryotic lineages than previously thought. The uniquely far red-shifted absorption spectra of the subset we designate RubyACRs provide the long-sought inhibitory optogenetic tools producing large passive currents activated by long-wavelength light, enabling deep tissue penetration. Previously only low-efficiency ion-pumping rhodopsins were available for neural inhibition by the orange-red region of the spectrum. The unusual amino acid composition of the retinal-binding pocket in RubyACRs expands our understanding of color tuning in retinylidene proteins. Finally, energy transfer from the fluorescent protein used as a tag on RubyACRs opens a potential new dimension in molecular engineering of optogenetic tools.

Theoretical Analysis of Hydrogen Bonding in the Active Site of Bovine Rhodopsin

The retinal binding pocket of bovine rhodopsin contains an extended hydrogen-bonded network that involves protein amino acids and water molecules. The protonation state and the role of Glu181, which is part of the hydrogen-bonded network, have been debated. According to the counterion switch model, Glu181 is protonated in the rhodopsin state and it becomes negatively charged (and a counterion for the protonated retinal Schiff base) in Meta II, upon proton transfer to Glu113[24, 25]. In contrast, in the complex counterion model Glu181 is negatively charged in rhodopsin, and the role of counterion is gradually shifted from Glu113 to Glu181 during activation [13]. Here we perform computer simulations to examine the energetics of a putative proton transfer path from Glu181 to the counterion of the retinal Schiff base, Glu113, in the rhodopsin and bathorhodopsin intermediates. The calculated energy barriers and reaction energies are significant. This suggests that proton transfer from Glu181 to Glu113 is very unlikely in the rhodopsin and bathorohodopsin protein structures.