title: Photochemical reaction cycle transitions during anion channelrhodopsin gating.
abstract: A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flash-induced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L⇔M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry & Molecular Biology, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030.
authors.forename: Oleg A
authors.initials: OA
authors.lastname: Sineshchekov
authors.affiliation: Center for Membrane Biology, Department of Biochemistry & Molecular Biology, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030.
authors.forename: Hai
authors.initials: H
authors.lastname: Li
authors.affiliation: Center for Membrane Biology, Department of Biochemistry & Molecular Biology, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030.
authors.forename: Elena G
authors.initials: EG
authors.lastname: Govorunova
authors.affiliation: Center for Membrane Biology, Department of Biochemistry & Molecular Biology, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030 john.l.spudich@uth.tmc.edu.
authors.forename: John L
authors.initials: JL
authors.lastname: Spudich
mesh_terms: D000838:Anions; D015640:Ion Channel Gating; D055668:Photochemical Processes; D012243:Rhodopsin / Q000502:physiology*
chemical_list: D000838:Anions; D012243:Rhodopsin
keywords: Schiff base; channel gating; channelrhodopsins; optogenetics; photochemical conversions