Proton transfer reactions in the red light-activatable channelrhodopsin variant ReaChR and their relevance for its function

• Published in: The Journal of biological chemistry 2017-08, Vol.292 (34), p.14205-14216
• Main Authors: Kaufmann, Joel C.D., Krause, Benjamin S., Grimm, Christiane, Ritter, Eglof, Hegemann, Peter, Bartl, Franz J.
• Format: Article
• Language: English
• Subjects: Algal Proteins - chemistry; Algal Proteins - genetics; Algal Proteins - metabolism; Amino Acid Substitution; Catalytic Domain - radiation effects; channelrhodopsin; Chlamydomonas reinhardtii - metabolism; Chlamydomonas reinhardtii - radiation effects; Chlorophyta - metabolism; Chlorophyta - radiation effects; Electrophysiological Phenomena; electrophysiology; Fourier transform IR (FTIR); gating; HEK293 Cells; Humans; Hydrogen Bonding - radiation effects; ion channel; Light; Models, Molecular; Molecular Biophysics; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutation; photobiology; photocycle; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Protein Conformation - radiation effects; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Stability; proton dynamics; proton transfer; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Rhodopsin - chemistry; Rhodopsin - genetics; Rhodopsin - metabolism; Spectroscopy, Fourier Transform Infrared; ultraviolet-visible spectroscopy (UV-vis spectroscopy)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.779629

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 Volvox carteri ChR1 red-activatable channelrhodopsin (“ReaChR,” λmax = 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 versus cations, such as Na+, K+, and Ca2+ (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 Glu130 (Glu90 in Chlamydomonas reinhardtii (Cr) 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 Asp293 (Asp253 in CrChR2) 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 Asp196 (Asp156 in CrChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15-syn-retinal at the expense of 15-anti, 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-anti species, whereas 15-syn species contribute only to the stationary current.