Proton transfer pathway in anion channelrhodopsin-1

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here, we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths was also analyzed using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shorten and lengthen the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild-type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients toward the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

Proton-mediated gating mechanism of anion channelrhodopsin-1

Anion channelrhodopsin from Guillardia theta (GtACR1) has Asp234 (3.2 Å) and Glu68 (5.3 Å) near the protonated Schiff base. Here we investigate mutant GtACR1s (e.g., E68Q/D234N) expressed in HEK293 cells. The influence of the acidic residues on the absorption wavelengths were also analyzed, using a quantum mechanical/molecular mechanical approach. The calculated protonation pattern indicates that Asp234 is deprotonated and Glu68 is protonated in the original crystal structures. The D234E mutation and the E68Q/D234N mutation shortens and lengthens the measured and calculated absorption wavelengths, respectively, which suggests that Asp234 is deprotonated in the wild type GtACR1. Molecular dynamics simulations show that upon mutation of deprotonated Asp234 to asparagine, deprotonated Glu68 reorients towards the Schiff base and the calculated absorption wavelength remains unchanged. The formation of the proton transfer pathway via Asp234 toward Glu68 and the disconnection of the anion conducting channel are likely a basis of the gating mechanism.

Time-resolved spectroscopic and electrophysiological data reveal insights in the gating mechanism of anion channelrhodopsin

Channelrhodopsins are widely used in optogenetic applications. High photocurrents and low current inactivation levels are desirable. Two parallel photocycles evoked by different retinal conformations cause cation-conducting channelrhodopsin-2 (CrChR2) inactivation: one with efficient conductivity; one with low conductivity. Given the longer half-life of the low conducting photocycle intermediates, which accumulate under continuous illumination, resulting in a largely reduced photocurrent. Here, we demonstrate that for channelrhodopsin-1 of the cryptophyte Guillardia theta (GtACR1), the highly conducting C = N-anti-photocycle was the sole operating cycle using time-resolved step-scan FTIR spectroscopy. The correlation between our spectroscopic measurements and previously reported electrophysiological data provides insights into molecular gating mechanisms and their role in the characteristic high photocurrents. The mechanistic importance of the central constriction site amino acid Glu-68 is also shown. We propose that canceling out the poorly conducting photocycle avoids the inactivation observed in CrChR2, and anticipate that this discovery will advance the development of optimized optogenetic tools.

Expression analysis of microbial rhodopsin-like genes in Guillardia theta

The Cryptomonad Guillardia theta has 42 genes encoding microbial rhodopsin-like proteins in their genomes. Light-driven ion-pump activity has been reported for some rhodopsins based on heterologous E. coli or mammalian cell expression systems. However, neither their physiological roles nor the expression of those genes in native cells are known. To reveal their physiological roles, we investigated the expression patterns of these genes under various growth conditions. Nitrogen (N) deficiency induced color change in exponentially growing G. theta cells from brown to green. The 29 rhodopsin-like genes were expressed in native cells. We found that the expression of 6 genes was induced under N depletion, while that of another 6 genes was reduced under N depletion.

The photoactive site modulates current rectification and channel closing in the natural anion channelrhodopsin GtACR1

ABSTRACTThe crystal structure of GtACR1 from Guillardia theta revealed an intramolecular tunnel predicted to expand to form the anion-conducting channel upon photoactivation (Li et al. 2019). The location of the retinylidene photoactive site within the tunnel raised the question of whether, in addition to triggering channel opening by photoisomerization, the site also participates in later channel processes. Here we demonstrate the involvement of the photoactive site in chloride conductance and channel closing. Electrostatic perturbation of the photoactive retinylidene Schiff base region by glutamate substitutions alters the rectification of the photocurrent as well as channel closing kinetics. Substitutions on opposite sides of the photoactive site causes opposite changes, with channel closing kinetically correlated with Schiff base deprotonation, and the extent of these effects closely correlate with distance of the introduced glutamyl residue from the photoactive site.

Crystal structure of a natural light-gated anion channelrhodopsin

The anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

Crystal Structure of a Natural Light-Gated Anion Channelrhodopsin

ABSTRACTThe anion channelrhodopsin GtACR1 from the alga Guillardia theta is a potent neuron-inhibiting optogenetics tool. Presented here, its X-ray structure at 2.9 Å reveals a tunnel traversing the protein from its extracellular surface to a large cytoplasmic cavity. The tunnel is lined primarily by small polar and aliphatic residues essential for anion conductance. A disulfide-immobilized extracellular cap facilitates channel closing and the ion path is blocked mid-membrane by its photoactive retinylidene chromophore and further by a cytoplasmic side constriction. The structure also reveals a novel photoactive site configuration that maintains the retinylidene Schiff base protonated when the channel is open. These findings suggest a new channelrhodopsin mechanism, in which the Schiff base not only controls gating, but also serves as a direct mediator for anion flux.

Bacteriorhodopsin-like channelrhodopsins: Alternative mechanism for control of cation conductance

The recently discovered cation-conducting channelrhodopsins in cryptophyte algae are far more homologous to haloarchaeal rhodopsins, in particular the proton pump bacteriorhodopsin (BR), than to earlier known channelrhodopsins. They uniquely retain the two carboxylate residues that define the vectorial proton path in BR in which Asp-85 and Asp-96 serve as acceptor and donor, respectively, of the photoactive site Schiff base (SB) proton. Here we analyze laser flash-induced photocurrents and photochemical conversions in Guillardia theta cation channelrhodopsin 2 (GtCCR2) and its mutants. Our results reveal a model in which the GtCCR2 retinylidene SB chromophore rapidly deprotonates to the Asp-85 homolog, as in BR. Opening of the cytoplasmic channel to cations in GtCCR2 requires the Asp-96 homolog to be unprotonated, as has been proposed for the BR cytoplasmic channel for protons. However, reprotonation of the GtCCR2 SB occurs not from the Asp-96 homolog, but by proton return from the earlier protonated acceptor, preventing vectorial proton translocation across the membrane. In GtCCR2, deprotonation of the Asp-96 homolog is required for cation channel opening and occurs >10-fold faster than reprotonation of the SB, which temporally correlates with channel closing. Hence in GtCCR2, cation channel gating is tightly coupled to intramolecular proton transfers involving the same residues that define the vectorial proton path in BR.