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 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.

Structural basis for ion selectivity in potassium-selective channelrhodopsins

“…Both Hc KCR1 and 2 form a trimer (Figures 1A and 1B), as was also observed in ChRmine, the only PLCR for which high-resolution structural information is available (Kishi et al, 2022; Tucker et al, 2022). The trimerization is mainly achieved by the direct and lipid-mediated interactions among transmembrane helices (TMs) 1-2 and TMs 4-5 of adjacent protomer, and the center of the trimer interface is filled with six lipid molecules (Figures 1A and 1B).…”

“…The C-terminal TM7 helix is also ∼1.5 turns longer than that of ChRmine (Figure 1F), making it more similar to that of canonical CCRs such as C1C2 (the chimera derived from Cr ChR1 and Cr ChR2) (Figure 1G). In PLCRs, residues from TM1, 2, 3, 7, and ECL1 form the core of the ion-conducting pathway within each monomer (Kishi et al, 2022; Tucker et al, 2022), so the structural differences of TM1, 7, and ECL1 observed in Hc KCRs change the shape of the pathway, to be discussed in more details later.…”

“…The residues comprising the retinal binding pocket are very similar between Hc KCR1, 2, and ChRmine (Figure 3A); 12 of 18 residues are conserved between Hc KCRs and ChRmine, and only two residues are different between Hc KCR1 and 2 (Figure 3B). To understand the function of these residues, we first introduced mutations to Y106 and T109 in Hc KCRs (Y116 and T119 in ChRmine) because a previous study showed that Y116W and T119A mutations in ChRmine significantly decelerate and accelerate off kinetics, respectively (Tucker et al, 2022). However, we found that the effects of corresponding mutations in Hc KCRs are very different; the Y106W mutation moderately decelerates the off kinetics of only Hc KCR2, and the T109A mutation does not accelerate but decelerates the off kinetics of only Hc KCR1 (Figures 3C, 3D, S5, S6A, and S6B).…”

Structural foundations of potassium selectivity in channelrhodopsins

“…An inter-protomer conductance has been proposed in ChRmine, based on the cryo-EM structure obtained in detergent (14). However, lipids block the space between the protomers in a ChRmine trimer incorporated in membranous nanodisks (15), which is expected to represent a state of the protein close to that in biological membranes.…”

“…KCR protein sequences show the highest homology to cryptophyte BCCRs out of all currently known ChRs (10), although their source organism is phylogenetically very distant from cryptophytes. High-resolution structures of only one BCCR, known as ChRmine, have been reported (14, 15). They show trimeric organization typical of haloarchaeal ion-pumping rhodopsins (16), whereas chlorophyte CCRs and cryptophyte ACRs form dimers (17, 18).…”