Retinal isomerization and water-pore formation in channelrhodopsin-2

Abstract

Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-µs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P₂³⁹⁰-like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening.

Keywords: ChR2; QM/MM protein modeling; channelrhodopsin-2; molecular dynamics; optogenetics.

Fig. 1.

Schematic representation of the intermediate states of the ChR2 photocycle and the respective protein conformational and hydration changes studied in the MD simulations.

Fig. 2.

Photocycle intermediates. (A) The D⁴⁷⁰ state displays the RSB H-bonding with E123. (B) All-trans to 13-cis isomerization of the retinal yields the K state (P₁⁵⁰⁰). The RSB is pointing toward D253. E90 and E123 interact via an H bond, and a water pore is formed (indicated by a purple dashed arrow). (C) The M state (P₂³⁹⁰) forms upon proton transfer from the RSB to D253. The retinal is in a planar conformation, with the nitrogen atom pointing toward the cytosolic side of the membrane. E90 is H-bonding with E123 and D253, in a hydrated central gate. W223 is displaced from its position in the dark state due to steric interactions with the C20 methyl group. The protein backbone is shown in ribbons. The yellow arrows indicate the side-chain conformational changes. H-bonding interactions are shown as dashed green lines.

Fig. 3.

Differential absorbance changes of ChR2 K- and M-state photointermediates from simulation (purple and blue lines, respectively) and experiment (orange and red lines, respectively) (10). The experimental curves are shifted by 42 and 75 nm, and the intensities are scaled by 0.7 and 1.05 and shifted by 0.12 and 0.15 units for the K and M states, respectively.

Fig. 4.

Water density (in red) of (A) a snapshot of the D⁴⁷⁰ structure, which presents a gap in the center of the channel near the retinal, K257, N258, and E90 (shown in licorice), and (B) a snapshot of the water pore after photoisomerization of the retinal. The head groups of the lipids are shown as tan spheres, and one of the chains of ChR2 is shown as transparent blue cartoon. The rest of the system is omitted for clarity.

Fig. 5.

Water preopen pore in the M state. (A) Connectivity of the water molecules. Lime bonds connect water molecules (red) within 4-Å distance. (B) Trajectory of a water permeation event. The oxygen atom of the water molecule is represented as a sphere colored according to the permeation time (as indicated by the color bar).