title: Complex Photochemistry within the Green-Absorbing Channelrhodopsin ReaChR.
abstract: Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (λmax= 532 nm) with a non-rhodopsin-like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (λmax= 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C15=N isomers of the retinal cofactor with either syn- or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M- (M2) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C13=C14, C15=N double-isomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C13=C14double-bond isomerization.
authors.affiliation: Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany. Electronic address: benjamin.krause@biologie.hu-berlin.de.
authors.forename: Benjamin S
authors.initials: BS
authors.lastname: Krause
authors.affiliation: Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Grimm
authors.affiliation: Institute for Medical Physics and Biophysics, Charité Berlin, Berlin, Germany.
authors.forename: Joel C D
authors.initials: JCD
authors.lastname: Kaufmann
authors.affiliation: Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Schneider
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, Rockefeller University, New York, New York.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.affiliation: Institute for Medical Physics and Biophysics, Charité Berlin, Berlin, Germany.
authors.forename: Franz J
authors.initials: FJ
authors.lastname: Bartl
authors.affiliation: Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Berlin, Germany. Electronic address: hegemann@rz.hu-berlin.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Hegemann
mesh_terms: D065638:Absorption, Radiation; D003116:Color; D057809:HEK293 Cells; D006801:Humans; D055668:Photochemical Processes; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D012243:Rhodopsin