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Abstract

The discovery of channelrhodopsins introduced a new class of ion channels whose conductance can be remotely controlled by light, the feature that founded optogenetics. To explore the connection between the gating mechanism and the influx and efflux of water molecules in channelrhodopsin-2 (ChR2), we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes that report on the hydration of transmembrane α-helices. We show that D253 accepts the proton released by the Schiff base (_τ1/2= 10 μs), the latter being reprotonated by D156 (_τ1/2= 2 ms) which is part of the DC gate. Previous conclusions on the involvement of E90 in channel opening are ruled out by demonstrating that E90 deprotonates exclusively in the non-conductive P⁴₄₈₀ state. Our results merge into a mechanistic proposal that relates the observed proton transfer reactions, protein conformational changes and backbone hydration to the gating of the cation channel. Our results will not only contribute to improve the properties of this optogenetic tool but will also help in elucidating the temporal sequences of ion channeling across the cellular membrane