title: Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating.
abstract: 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(2)(390) and P(3)(520)) 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(4)(480) 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.
authors.affiliation: Experimental Molecular Biophysics, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: Víctor A
authors.initials: VA
authors.lastname: Lórenz-Fonfría
authors.forename: Tom
authors.initials: T
authors.lastname: Resler
authors.forename: Nils
authors.initials: N
authors.lastname: Krause
authors.forename: Melanie
authors.initials: M
authors.lastname: Nack
authors.forename: Michael
authors.initials: M
authors.lastname: Gossing
authors.forename: Gabriele
authors.initials: G
authors.lastname: Fischer von Mollard
authors.forename: Christian
authors.initials: C
authors.lastname: Bamann
authors.forename: Ernst
authors.initials: E
authors.lastname: Bamberg
authors.forename: Ramona
authors.initials: R
authors.lastname: Schlesinger
authors.forename: Joachim
authors.initials: J
authors.lastname: Heberle
mesh_terms: D000075402:Channelrhodopsins; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology* / Q000528:radiation effects; D007700:Kinetics; D007834:Lasers; D008956:Models, Chemical; D008958:Models, Molecular; D010777:Photochemistry; D011487:Protein Conformation; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000075402:Channelrhodopsins; D011522:Protons