Photoactivation of channelrhodopsin

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, D-470/D-480. Laser flashes converted both with tau approximate to 200 mu s into major photointermediates P-510/P-530, which reverted back to the dark states with tau approximate to 15-100 ms. Both intermediates were assigned to conducting states. Three early intermediates P-500/P-515 and P-390 were detected on a ns to mu s 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.