The discovery of channelrhodopsins introduced a new class of lightgated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, 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 at 1,665(â) and 1,648(+) cm. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 ÎŒs (30%) and 200 ÎŒs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.channelrhodopsin | optogenetics | channel gating | infrared spectroscopy | time-resolved spectroscopy I on channels are membrane proteins that mediate the passive movement of cations and anions across biological membranes, a process not only central for most living organisms but key to electrical excitability of cells. Upon activation, ion channels constitute a transient pathway to facilitate the permeation of ions across the cellular membrane. Ion channels can be switched, or gated, from a closed (nonconductive) to an open (conductive) state by external stimuli, such as ligands, voltage, or mechanical stress (1-3). Available structural information indicates that the ion-conducting pathway generally comprises a pore with wide regions solvated by water and constrictions sites formed by specific polar groups that confer ion selectivity (4). In the nonconductive state, the permeation of ions is prevented by energy barriers along the pore, either from physical occlusion or from the hydrophobic nature of residues lining the pore: the pore does not need to be physically occluded to be functionally closed (5). Habitual suspects for the gating mechanism, i.e., how the protein transits from the nonconductive to the conductive state, are diverse types of structural changes, such as orientation/rotation changes of transmembrane helices or of bulky (aromatic) side chains (6). Several computational studies have suggested a more subtle gating mechanism, where the thermodynamics and kinetics of the hydration of the ion-conducting pore might play a vital role (5,7,8). However, experimental support for this last proposal relies largely on studies on synthetic nanometric pores (9).Channelrhodopsins (ChRs) belong to the new class of lightgated ion channels, i.e., ion channels activated by light...