期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：43
页码：E5796-E5804
DOI：10.1073/pnas.1511462112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe passive movement of ions across biological membranes is controlled by channels. How these membrane proteins are activated and become permeable to ions, a process with high biomedical and biotechnological impact, has been the subject of numerous structural, functional, and computational studies. We have investigated the light-gated cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii, widely used in optogenetics, by combining time-resolved infrared spectroscopy and electrophysiology. The temporal evolution of the hydration of transmembrane -helices, identified by spectroscopic markers, matches the kinetics of ion conductance, as monitored by electrophysiology. Our results provide a solid experimental ground for previous computational studies suggesting that the thermodynamics and kinetics of hydration of the ion-conducting pore are key aspects to understand the regulation of ion channels. The discovery of channelrhodopsins introduced a new class of light-gated 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(-) and 1,648(+) cm-1. These two bands report on the hydration of transmembrane -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 s (30%) and 200 s (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.
