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, 112 (6), 1166-1175

Complex Photochemistry Within the Green-Absorbing Channelrhodopsin ReaChR

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Complex Photochemistry Within the Green-Absorbing Channelrhodopsin ReaChR

Benjamin S Krause et al. Biophys J.

Abstract

Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (λmax = 532 nm) with a non-rhodopsin-like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (λmax = 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C15=N isomers of the retinal cofactor with either syn- or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M- (M2) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C13=C14, C15=N double-isomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C13=C14 double-bond isomerization.

Figures

Figure 1
Figure 1
Photocurrent properties of ReaChR in HEK293 cells. (A) Helix architecture of the chimeric ReaChR consisting of VcChR1, helix 6 (H6) of VcChR2, and the N-terminal sequence of CrChR1. H6 of VcChR1 and VcChR2 only differ in five amino acids. (B) Action spectra (10 ms activation) of ReaChR at different external pH (pHe) values (mean ± SE, n = 7–12). (C) Light titration curve after excitation with green (530 nm; intensities from 6.03 × 10−5 mW mm−2 (0.01%) to 0.61 mW mm−2 (100%)) or orange light (600 nm; 6.65 × 10−5 to 0.47 mW mm−2); photocurrents are normalized to the highest peak response of each cell (mean ± SE, n = 6), lines were added for visual guidance. (D) Comparison of photocurrents evoked with 530 and 600 nm at the same cell (equal photon flux, n = 6). (E) Stationary action spectra at 1, 5, 50 and 100% light intensity (left, mean ± SE, n = 5–6). At pH 5.0 (red bars, right), reduction of the stationary current is more pronounced at 530 nm compared to 620 nm, implying that the photoreactive intermediate is accumulated more at pH 5.0 (mean ± SE, n = 6–8).
Figure 2
Figure 2
Spectral properties of recombinant ReaChR. (A) IDA spectra of purified ReaChR at pH 5.0, 7.4, and 9.0 (normalized to 280 nm). (B) Transient absorption changes induced by green laser flashes (10 ns, 530 nm, 5 mJ) were measured in saline detergent (0.03% DDM) solution at pH 5.0 (top, 100 mM NaCl, 10 mM citric acid), 7.4 (middle, DPBS), and 9.0 (bottom, 100 mM NaCl, 10 mM TRIS). Half-life times (t1/2) are highlighted in the contour plots. Five photocycle-intermediates K, L, M, N (not seen at pH 7.4), and O were identified. L and M are enriched at pH 5 and 9, respectively. Acidic pH decelerates photocycle kinetics. (C) Calculated absolute spectra of the L- and N-states of ReaChR at pH 5. Dots resemble raw data points and solid lines were added for visual guidance. (D) Cryostatic UV-vis measurements (light-dark, pH 7.4) of ReaChR under continuous illumination reveal maximal accumulation of the K- and L-states at 150 and 240 K, respectively. (E) FTIR difference (light-dark) of the photostationary states under continuous illumination at 150 (orange) and 240 K (blue) at pH 7.4. Imposing HOOP vibration at 968(+) cm−1 points toward a distorted 13-cis chromophore in the K-state, while cofactor strain is almost completely relieved in the upcoming L-state.
Figure 3
Figure 3
Photoconversion of intermediate states and photocycle model. (A) Absorption spectra of ReaChR-C168S recorded for the IDA and after excitation (60 s, 530 nm). Difference spectra were calculated (light-dark, top inset). Upon prolonged illumination, M-state (λmax = 396 nm) is accumulated predominantly, which completely decays within 90 min, but substantial absorption at ∼520 nm (∼55%) indicates the presence of another green-absorbing intermediate (N-state). Chromophore bleaching monitored at 550 nm involves two processes (bottom inset), whereby the faster component is attributed to M-state formation (1→2) and the slower one to formation of minor blue-shifted species seen at 420 and 460 nm (P420/460) (2→3). (B) Photocurrents of HEK cells expressing ReaChR-C168A under continuous illumination with green light and additional stronger light pulses (380–670 nm) inducing current inactivation. The 400 nm light effectively inactivates currents with slow recovery, whereas inactivation at 600 nm is smaller and instantaneously reversible with green light. (C) These current traces show more clearly that application of a single blue-light pulse on top of green background light causes current inactivation and slow recovery, whereas a 600 nm light pulse causes smaller inactivation but fast recovery. (D) Recombinant ReaChR-C168S illuminated with green (530 nm, weak) and/or blue (400 nm, strong) light, and rise and decay of the M- (380 nm) and dark states (550 nm) were monitored by single kinetic traces. Green-light illumination induces formation of M and dark state (D) bleaching in parallel, while blue light reverses this process, indicative for an M→D shortcut. (E) Proposed symmetrical two-cycle model of ReaChR. One cycle is based on the 13-trans, 15-anti retinal configuration (anti-cycle) while the other starts from 13-cis, 15-syn (syn-cycle); the equilibrium of both forms the apparent dark state (DAapp, gray background), while the IDA is supposed to consist of 100% all-trans retinal. Upon illumination with green light, D converts to a red-shifted K-like intermediate, succeeded by the blue-shifted states L and M. The deprotonated M-state decays biexponentially (M1 and M2) and is reprotonated during N-state formation. N decays to an intermediate that is spectrally not distinguishable from the dark state (O-state). (Colored arrows) Proposed photoinduced pathways between the intermediates and both cycles; (black arrows) thermal conversions. The conducting states are assigned to the M2- and N-equilibrium (red box). The syn-cycle consists of the same spectral states marked by a hyphen and undergoes the same protonations; corresponding shortcuts and the M-substates are omitted for clarity. Upon prolonged illumination of slow-cycling mutants, side-products (P420/460) are accumulated, which could be photoconverted by UV-light (data not shown).

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