Chloride conducting channelrhodopsins (ChloCs) are new members of the optogenetic toolbox that enable neuronal inhibition in target cells. Originally, ChloCs have been engineered from cation conducting channelrhodopsins (ChRs), and later identified in a cryptophyte alga genome. We noticed that the sequence of a previously described Proteomonas sulcata ChR (PsChR1) was highly homologous to the naturally occurring and previously reported ChloCs GtACR1/2, but was not recognized as an anion conducting channel. Based on electrophysiological measurements obtained under various ionic conditions, we concluded that the PsChR1 photocurrent at physiological conditions is strongly inward rectifying and predominantly carried by chloride. The maximum activation was noted at excitation with light of 540 nm. An initial spectroscopic characterization of purified protein revealed that the photocycle and the transport mechanism of PsChR1 differ significantly from cation conducting ChRs. Hence, we concluded that PsChR1 is an anion conducting ChR, now renamed PsACR1, with a red-shifted absorption suited for multicolor optogenetic experiments in combination with blue light absorbing cation conducting ChRs.

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 (λ = 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 (λ = 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 C=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- (M) 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 C=C, C=N double-isomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C=C double-bond isomerization.

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. ChR variants with a red-shifted absorption maximum, such as the modified ChR1 red-activatable channelrhodopsin ("ReaChR," λ = 527 nm), are of particular interest because longer wavelengths allow optical excitation of cells in deeper layers of organic tissue. In all ChRs investigated so far, proton transfer reactions and hydrogen bond changes are crucial for the formation of the ion-conducting pore and the selectivity for protons cations, such as Na, K, and Ca (1). By using a combination of electrophysiological measurements and UV-visible and FTIR spectroscopy, we characterized the proton transfer events in the photocycle of ReaChR and describe their relevance for its function. 1) The central gate residue Glu (Glu in () ChR2) (i) undergoes a hydrogen bond change in D → K transition and (ii) deprotonates in K → M transition. Its negative charge in the open state is decisive for proton selectivity. 2) The counter-ion Asp (Asp in ChR2) receives the retinal Schiff base proton during M-state formation. Starting from M, a photocycle branching occurs involving (i) a direct M → D transition and (ii) formation of late photointermediates N and O. 3) The DC pair residue Asp (Asp in ChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15--retinal at the expense of 15-, which is the predominant isomer in the wild type, and abolishes the peak current in electrophysiological measurements. This suggests that the peak current is formed by 15- species, whereas 15- species contribute only to the stationary current.

Anion channelrhodopsins (ACRs) are of great interest due to their ability to inhibit electrical signaling in optogenetic experiments. The photochemistry of ACRs is currently poorly understood and an improved understanding would be beneficial for rational design of ACRs with modified properties. Activation/deactivation of ACRs involves a series of photoreactions ranging from femtoseconds to seconds, thus real-time observation is essential to comprehend the full complexity of the photochemical processes. Here we investigate the photocycle of an ACR from Proteomonas sulcata (PsACR1), which is valuable for optogenetic applications due to the red-shifted absorption and action spectra compared to the prototype ACRs from Guillardia theta: GtACR1 and GtACR2, and the fast channel closing properties. From femto-to-submillisecond transient absorption spectroscopy, flash photolysis, and point mutations of acidic residues near the retinal Schiff base (RSB), E64, and D230, we found that the photoisomerization occurs in ∼500 fs independent of the protonation state of E64. Notably, E64 is involved in the rearrangement of the hydrogen-bond network near the RSB after photoisomerization. Furthermore, we suggest that E64 works as a primary proton acceptor during deprotonation of the RSB as has been proposed for GtACR1. Our findings allow for a deeper understanding of the photochemistry on the activation/deactivation of ACRs.

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Channelrhodopsins (ChRs) are retinal binding membrane proteins found in single-cell algae. Photoisomerization of ChRs leads to formation of an ion channel. The resulting change in membrane voltage modulates flagellate motions allowing phototaxis and photophobic responses. Heterologously expressed in host cells, ChRs allow the evocation or suppression of changes in membrane potential with high spatio-temporal resolution -this method has become known as optogenetics. Functional studies have raised questions concerning the molecular determinants for absorption, formation and closing of the ion channel and ion selectivity. It was the scope of this thesis to address these questions based on a comparison of the three different ChR variants C1C2, ReaChR (Red-activatable ChR) and Chrimson. C1C2 (λmax ≈ 470 nm) is a chimera of the Chlamydomonas reinhardtii ChRs CrChR1 and CrChR2. ReaChR (λmax ≈ 520 nm) is a variant of Volvox carteri ChR1 whose red-shifted absorption allows its use in deeper layers of organic tissue in optogenetic experiments. The even further red-shifted (Cs)Chrimson (λmax ≈ 590 nm) is a more distantly related ChR from Chlamydomonas noctigama with the N-terminal sequence from Chloromonas subdivisa ChR that is significantly more proton-selective. The photoreaction mechanism was investigated using FTIR (Fourier Transform Infrared) spectroscopy at room temperature and at cryostatic conditions. The results were complemented by UV-Vis spectroscopy and retinal extraction and subsequent HPLC (High Performance Liquid Chromatography) analysis.As in most microbial rhodopsins, the retinal cofactor in ChRs is predominantly in 13-trans,15anti conformation and bound to the protein by a retinal Schiff base (RSB) linkage to a lysine.Usually, the RSB is protonated in the dark (RSBH + ) stabilized by the counter-ion complex formed by a glutamate (counter-ion 1, Ci1) and an aspartate (counter-ion 2, Ci2).Photoreceptors are optimized to use photon energy to drive conformational changes of the protein backbone. Therefore, a fraction of the photon energy is stored by a transient distortion of the chromophore and separation of the charges in the active site by increased distance between the RSBH + and its counter-ions. In this thesis, it is shown that in ReaChR the transfer of the stored energy to the protein is largely affected by the Ci1 (Glu163) protonation state, being decelerated by protonated Ci1 due to an enhanced rigidity of the active site that stabilizes the distorted chromophore conformation. Instead, in Chrimson the chromophore

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The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH þ ) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH þ -counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH þ . Ci1 retains a pK a of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogenbonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.