Abstract
ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
Highlights
Light—a crucial energy source and environmental signal—is typically captured by motile organisms using rhodopsins, which are largely classified into two groups: microbial and animal, both consisting of a seven-transmembrane (7TM) protein and a covalently bound chromophore
The fundamental limitation of single-particle cryo-electron microscopy (cryo-EM) is that images of small membrane proteins in detergent micelles have insufficient features for image alignment in data processing
The density was of excellent quality, allowing accurate modeling of ChRmine continuously from residues 10 to 279, excluding the disordered N-terminal nine residues and C-terminal 25 residues (Figures S2K–S2R; STAR Methods), and clearly resolved several lipids, water molecules, and the retinal
Summary
Light—a crucial energy source and environmental signal—is typically captured by motile organisms using rhodopsins, which are largely classified into two groups: microbial and animal, both consisting of a seven-transmembrane (7TM) protein (opsin) and a covalently bound chromophore (retinal). Light absorption induces retinal isomerization followed by photocycle, a series of photochemical reactions (Zhang et al, 2011; Ernst et al, 2014; Deisseroth and Hegemann, 2017), which in microbial rhodopsins exerts direct biochemical action (examples include pumps, channels, sensors, and enzymes) (Kandori, 2020; Kato, 2021) Targeted expression of these proteins (especially of the channel- and pump-type) in specific cell types, when applied along with precise light delivery, enables causal study of cellular activity in behaving organisms (optogenetics) (Deisseroth, 2015, 2021; Kurihara and Sudo, 2015).
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