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.

Download Full-text

Crystal Structure of a Natural Light-Gated Anion Channelrhodopsin

10.1101/405308 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hai Li ◽

Chia-Ying Huang ◽

Elena G. Govorunova ◽

Christopher T. Schafer ◽

Oleg A. Sineshchekov ◽

...

Keyword(s):

Crystal Structure ◽

Schiff Base ◽

Natural Light ◽

X Ray ◽

Anion Conductance ◽

Guillardia Theta ◽

Cytoplasmic Side

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.

Download Full-text

Crystal structure of a natural light-gated anion channelrhodopsin

eLife ◽

10.7554/elife.41741 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

Author(s):

Hai Li ◽

Chia-Ying Huang ◽

Elena G Govorunova ◽

Christopher T Schafer ◽

Oleg A Sineshchekov ◽

...

Keyword(s):

Crystal Structure ◽

Schiff Base ◽

Natural Light ◽

X Ray ◽

Anion Conductance ◽

Guillardia Theta ◽

Cytoplasmic Side

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.

Download Full-text

Proton-mediated gating mechanism of anion channelrhodopsin-1

10.1101/2021.07.22.453395 ◽

2021 ◽

Author(s):

Masaki Tsujimura ◽

Keiichi Kojima ◽

Shiho Kawanishi ◽

Yuki Sudo ◽

Hiroshi Ishikita

Keyword(s):

Schiff Base ◽

Hek293 Cells ◽

Wild Type ◽

Conducting Channel ◽

Gating Mechanism ◽

Guillardia Theta ◽

In The Wild ◽

Mechanical Approach ◽

Dynamics Simulations ◽

Original Crystal

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.

Download Full-text