Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs

Abstract

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 broad, red-shifted activation spectrum, cation selectivity, and large photocurrents, while its slow closing kinetics impedes 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 channel properties could be tuned with mutations and we identify ChRmine variants with ten-fold decreased and two-fold increased closing rates. A T119A mutant shows favorable properties relative to wild-type and previously reported ChRmine variants for optogenetics. 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 experiments.

WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

“…The identified K + filter contrasts the conserved mechanism of ion selectivity in conventional highly symmetric tetrameric K + channels, like KcsA or K v , where K + ions are conducted along the tetramer interface and are tightly coordinated by backbone carbonyl oxygens of the selectivity filter ( 24 ). However, for KCRs, our equilibrated structural models and the results of our mutational analysis predict a trimeric assembly with an inter-subunit cavity most likely filled with lipids similar to ChRmine ( 19 ) and an ion permeation pathway going through the individual KCR protomers themselves. We propose that the passage of K + ions requires dehydration to pass the hydrophobic and aromatic side chains of the K + selectivity filter where passage of Na + —with higher dehydration energy and more localized charge—is blocked.…”

“…Both KCRs have a bacteriorhodopsin-like DTD motif in membrane helix 3 (D85, T89, and D96 in bacteriorhodopsin). This is a feature shared with the related cryptophyte CCRs that were first discovered in Guillardia theta ( 18 ) and that include the green light–activated nonselective cation channel ChRmine from Rhodomonas lens , the structure of which was recently solved by cryo–electron microscopy ( 19 , 20 ). Despite a thorough initial characterization by Govorunova and colleagues ( 17 ), the molecular determinants of K + selectivity in this just recently identified group of KCRs remain unknown.…”

“…On the basis of the sequence similarity to ChRmine, a trimeric assembly was chosen. For ChRmine, the trimeric cryo–electron microscopy structure was used (PDB-ID 7SFK) ( 19 ). Each protomer of HcKCR1, WlChR1, and B1ChR2 was equipped with a retinal cofactor bound to K233 in HcKCR1, K251 in WlChR1, and K232 in B1ChR2.…”

Structural Foundations of Potassium Selectivity in Channelrhodopsins

“…KCR protein sequences show the highest homology to cryptophyte BCCRs out of all currently known ChRs ( 12 ), although their source organism is phylogenetically very distant from cryptophytes. High-resolution structures of only one BCCR, known as ChRmine, have been reported ( 16 , 17 ). They show trimeric organization typical of haloarchaeal ion-pumping rhodopsins ( 18 ), whereas chlorophyte CCRs and cryptophyte ACRs form dimers ( 19 , 20 ).…”

Cryo-EM structures of kalium channelrhodopsins KCRs

“…Overall, KCRs adopt trimer architectures similar to ChRmines 7,8 , instead of dimer formation 9,10 known for light activated non-selectivity cation channels (Fig. 1a-f).…”