Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating

• Published in: Proceedings of the National Academy of Sciences - PNAS 2013-04, Vol.110 (14), p.E1273-E1281
• Main Authors: Lórenz-Fonfría, Víctor A, Resler, Tom, Krause, Nils, Nack, Melanie, Gossing, Michael, Fischer von Mollard, Gabriele, Bamann, Christian, Bamberg, Ernst, Schlesinger, Ramona, Heberle, Joachim
• Format: Article
• Language: English
• Subjects: Bacteria; Biological Sciences; Cells; Channelrhodopsins; HEK293 Cells; Humans; Ion Channel Gating - physiology; Ion Channel Gating - radiation effects; Kinetics; Lasers; Measurement; Models, Chemical; Models, Molecular; Photochemistry; Physical Sciences; PNAS Plus; Protein Conformation; Proteins; Protons; Spectroscopy, Fourier Transform Infrared
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1219502110

The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements. We have resolved the vibrational changes associated with the open states of the channel (P ₂³⁹⁰ and P ₃⁵²⁰) and characterized several proton transfer events. Analysis of the amide I vibrations suggests a transient increase in hydration of transmembrane α-helices with a t ₁/₂ = 60 μs, which tallies with the onset of cation permeation. Aspartate 253 accepts the proton released by the Schiff base (t ₁/₂ = 10 μs), with the latter being reprotonated by aspartic acid 156 (t ₁/₂ = 2 ms). The internal proton acceptor and donor groups, corresponding to D212 and D115 in bacteriorhodopsin, are clearly different from other microbial rhodopsins, indicating that their spatial position in the protein was relocated during evolution. Previous conclusions on the involvement of glutamic acid 90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the nonconductive P ₄⁴⁸⁰ state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions and the protein conformational changes to the gating of the cation channel.