Photochemical Properties of the Red‐shifted Channelrhodopsin Chrimson

Abstract

Color-tuned variants of channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light-induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground-state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state.

Crystal structure of the red light-activated channelrhodopsin Chrimson

“…3e, f and Supplementary Figure 8a–c ). This can be explained by the decreased p K a and consequent deprotonation of the remaining counterion residue (Asp295 for the Glu165 mutants and Glu165 for the Asp295 mutants), consistent with previous studies of Chrimson 22 , 27 and other channelrhodopsins 29 . The blue-shift was more prominent when Asp295 is mutated, whereas the mutation of Glu165 to Gln or Ala had a moderate effect.…”

“…Therefore, the Glu165 proton might be partly shared by both counterion residues, via hydrogen bond interactions. Most importantly, the counterion complex is shielded from the extracellular bulk solvent by the hydrophobic side chain of Phe135, which is essential to stabilize the protonated form of the carboxylate, as reported in previous studies 22 , 27 , 29 . (Fig.…”

“…3a–d ). Recent studies have indicated that the highly red-shifted absorption of Chrimson is partly due to the protonation of the counterion residue Glu165 22 , 27 , which weakens the stabilization effect for the PSB, thus causing red-shifted absorption similar to that of the acidic form of BR (BR 605 ) 28 . Consistent with these studies, the purified Chrimson protein showed a large pH-dependent spectrum shift (Supplementary Figure 6a and b).…”

Cortical layer–specific critical dynamics triggering perception

“…We also tested the pH dependence of opsin-mediated genetically encoded calcium indicator (GECI) signals at external pH 7.0, 7.2, and 7.4. CsChrimson use was associated with pH-dependent Ca 2+ transients (29, 30). In contrast, no such effect was observed with ChRmine (fig.…”

Tracking Pore Hydration in Channelrhodopsin by Site-Directed Infrared-Active Azido Probes

“…49 Three mechanistic studies of the ReaChR photocycle were published with participation of the author. [51][52][53] (Cs)Chrimson is a construct of the natural red-shifted ChR (λmax ≈ 590 nm 39,54 ) discovered in Chlamydomonas noctigama by Klapoetke et al 2014 38 and the Nterminal fragment from Chloromonas subdivisa ChR 39 . The promising combination of a bathochromically shifted absorption and large photocurrents makes Chrimson an interesting target for mechanistic characterization.…”