Photoactivation of Channelrhodopsin

• Published in: The Journal of biological chemistry 2008-01, Vol.283 (3), p.1637-1643
• Main Authors: Ernst, Oliver P., Murcia, Pedro A. Sánchez, Daldrop, Peter, Tsunoda, Satoshi P., Kateriya, Suneel, Hegemann, Peter
• Format: Article
• Language: English
• Subjects: Animals; Electrodes; Electrophoresis, Polyacrylamide Gel; Ion Channel Gating - radiation effects; Light; Models, Biological; Oocytes; Rhodopsin - isolation & purification; Rhodopsin - metabolism; Spectrophotometry, Ultraviolet; Volvox - cytology; Volvox - metabolism; Volvox - radiation effects; Volvox carteri; Xenopus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M708039200

Channelrhodopsins (ChRs) are light-gated ion channels that control photomovement of microalgae. In optogenetics, ChRs are widely applied for light-triggering action potentials in cells, tissues, and living animals, yet the spectral properties and photocycle of ChR remain obscure. In this study, we cloned a ChR from the colonial alga Volvox carteri, VChR. After electrophysiological characterization in Xenopus oocytes, VChR was expressed in COS-1 cells and purified. Time-resolved UV-visible spectroscopy revealed a pH-dependent equilibrium of two dark species, D470/D480. Laser flashes converted both with τ ≈ 200 μs into major photointermediates P510/P530, which reverted back to the dark states with τ ≈ 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P500/P515 and P390 were detected on a ns to μs time scale. The spectroscopic and electrical data were unified in a photocycle model. The functional expression of VChR we report here paves the way toward a broader structure/function analysis of the recently identified class of light-gated ion channels.