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.
Ernst
Oliver P
OP
Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany. Electronic address: oliver.ernst@charite.de.
Murcia
Pedro A Sánchez
PAS
Institut für medizinische Physik und Biophysik, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Daldrop
Peter
P
Institut für Biologie, Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany.
Tsunoda
Satoshi P
SP
Institut für Biologie, Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany.
Kateriya
Suneel
S
Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India.
Hegemann
Peter
P
Institut für Biologie, Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin, Germany. Electronic address: hegemape@rz.hu-berlin.de.
D000818:Animals; D004566:Electrodes; D004591:Electrophoresis, Polyacrylamide Gel; D015640:Ion Channel Gating / Q000528:radiation effects*; D008027:Light; D008954:Models, Biological; D009865:Oocytes; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D044446:Volvox / Q000166:cytology / Q000378:metabolism* / Q000528:radiation effects*; D014981:Xenopus
D012243:Rhodopsin