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 approximately 200 mus into major photointermediates P(510)/P(530), which reverted back to the dark states with tau approximately 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 mus 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.