Light-dark adaptation of channelrhodopsin C128T mutant

Abstract

Channelrhodopsins are microbial type rhodopsins that operate as light-gated ion channels. Largely prolonged lifetimes of the conducting state of channelrhodopsin-2 may be achieved by mutations of crucial single amino acids, i.e. cysteine 128. Such mutants are of great scientific interest in the field of neurophysiology because they allow neurons to be switched on and off on demand (step function rhodopsins). Due to their slow photocycle, structural alterations of these proteins can be studied by vibrational spectroscopy in more detail than possible with wild type. Here, we present spectroscopic evidence that the photocycle of the C128T mutant involves three different dark-adapted states that are populated according to the wavelength and duration of the preceding illumination. Our results suggest an important role of multiphoton reactions and the previously described side reaction for dark state regeneration. Structural changes that cause formation and depletion of the assumed ion conducting state P520 are only small and follow larger changes that occur early and late in the photocycle, respectively. They require only minor structural rearrangements of amino acids near the retinal binding pocket and are triggered by all-trans/13-cis retinal isomerization, although additional isomerizations are also involved in the photocycle. We will discuss an extended photocycle model of this mutant on the basis of spectroscopic and electrophysiological data.

FIGURE 1.

Dark states and photostationary states of C128T channelrhodopsin. A, ChR2 mutant C128T, reconstituted in l-α-phosphatidylcholine vesicles in the dark, was illuminated 600 s by blue (470 nm) light. The spectral changes of optical density (OD) of the prominent 1661 cm⁻¹ band (as shown in the inset, blue line) are drawn as a function of time (black line). The illumination period is marked as a blue bar. We note the formation of the illuminated state (blue) immediately after the start of the illumination. It then evolves into a photostationary state (red). After the end of the illumination, the system converts via an intermediate state (light green) into a dark-adapted state (DAB, dark green). Underlined numbers indicate conversions used to calculate double difference spectra as shown in the next panel. B, infrared difference spectra photoproduct minus dark state of the illuminated state (measured immediately after start of illumination; blue), the photostationary state (red), the intermediate state (light green), and the dark-adapted state (DAB; green), achieved after 3600 s in the dark. For better comparison, the spectra were scaled. Double differences of the interconversions between these states were then calculated as indicated in A and numbered accordingly. C, upper trace: the same sample as shown in A was, after a regeneration period of 3 h after the blue illumination, exposed to a green illumination (300 s; 520 nm), and the intensity at 1661 cm⁻¹ was recorded as a function of time. After lights off, the sample evolved into a new dark-adapted state (DAG). A subsequently applied blue illumination of the sample initiated its return into DAG. Lower trace: flash illumination, either green or blue, caused the same effect as long green illumination, thereby finally leading to the DAG state (blue and green arrows). Extended blue illumination (blue bar) led back to DAB. D, FTIR and UV-visible (inset) difference spectrum (double difference) of DAB minus DAG (black line). We note differences in the structurally sensitive parts of the spectrum >1600 cm⁻¹ as well as in the region indicative for chromophore geometry (1150–1250 cm⁻¹). The double difference between the state induced by flash illumination and DAB is also given for comparison (gray line). abs., absorbance.

FIGURE 2.

Retinal isomers during the photocycle. A, retinal aldehyde HPLC extraction of different C128T states was performed at the times indicated after a 5-min blue or green illumination. Shown are the data from one single experiment. In the initial dark state, we found a mixture of 22% 13-cis and 78% all-trans, as reported previously. Surprisingly, after the extended illumination, we found significantly increased contributions of cis retinals. The share of 13-cis retinal was, however, dependent on illumination conditions; its maximum was reached 180 s after light. We found a slightly lesser maximal amount after a blue illumination (left column) than after a green illumination (right column). The relaxation into a dark state-like retinal mixture occurred on a very slow timescale that was even slower than the observed spectral alterations. B, extracted retinal isomer mixture before and at different times after the illumination; shown are 13-cis retinal (red), 11-cis retinal (green), 9-cis retinal (cyan), and all-trans retinal (blue). C, time course of the extracted retinal isomer mixture after application of a green (520 nm) instead of a blue illumination. Color codes are the same as described in B.

FIGURE 3.

The kinetic data of C128T during alternating green (520 nm) and blue (470 nm) illumination were analyzed by singular value decomposition and rotation procedure. A dark-adapted sample (DAB) was illuminated with blue (dark gray bar) and green (light gray bar) light alternatively. Each illumination was performed for 5 s, and then the wavelength of the incident light was immediately changed. Initial spectra were recorded with a time resolution of 200 ms. Subsequent data analysis yielded four main spectral components (b-spectra are scaled; absorbance is given in arbitrary units (a.u.); left column) that could be separated due to their kinetic behavior (right column). The first three components A–E, as seen from their kinetics B–F, reflect alterations of the protein induced once by the illumination but not directly dependent on the wavelength of incident light. Only the fourth component (G) closely followed the illumination procedure (H). abs., absorbance.

FIGURE 4.

Retinal isomers of retinylidene proteins. In the bacteriorhodopsin dark-adapted state, either all-trans, 15-anti or 13-cis, 15-syn retinal is observed, whereas 13-cis, 15-anti retinal occurs in the M intermediate structure. In bovine rhodopsin, the switch between all-trans, 15-anti and all-trans, 15-syn is capable to switch between the active form and its thermal predecessor.

FIGURE 5.

The proposed reaction scheme of ChR2-C128T. Black arrows label thermal reactions, blue and green arrows label reactions induced by blue (470 nm) and green (520 nm) light, respectively, and turquoise arrows label reactions that are triggered by both green and blue light. The proposed isomeric states of the retinal are given in gray. The three observed dark states IDA, DAB, and DAG are composed of only two dark species D470 and D480, which occur in different contributions due to the pretreatment of the sample. The unique IDA (from which the reaction starts) is mainly constituted of the species D480, only with minor contributions of D470. Similarly, D480 is the main component of DAB, whereas in DAG, D470 dominates. A photocycle is started by green or blue illumination from either D470 or D480. After the early intermediates (P500 and P500′, accordingly, not shown) and transient SB deprotonation (P390 and P390′), the conducting states P520 and P520′ are formed. The reaction proceeds further to a late intermediate (P480), which is equal or closely similar for both cycles. From this intermediate, D480 and D470 are reformed in a thermal reaction, and the pathway leading to D470 is favored. Alternatively, the side pathway with its intermediate P380 is populated either thermally or light-induced. Although thermal decay of P380 leads only into D470, its excitation by light enables formation of D480 via additional intermediates. Hence, another dark-adapted state (DAB) is only observed after long blue illumination. The D480 species can only be populated in significant amounts through the UV-shifted intermediates of the C128T side pathway (P380, P353, not shown) in a multiphoton reaction.