title: Intramolecular proton transfer in channelrhodopsins.
abstract: Channelrhodopsins serve as photoreceptors that control the motility behavior of green flagellate algae and act as light-gated ion channels when heterologously expressed in animal cells. Here, we report direct measurements of proton transfer from the retinylidene Schiff base in several channelrhodopsin variants expressed in HEK293 cells. A fast outward-directed current precedes the passive channel current that has the opposite direction at physiological holding potentials. This rapid charge movement occurs on the timescale of the M intermediate formation in microbial rhodopsins, including that for channelrhodopsin from Chlamydomonas augustae and its mutants, reported in this study. Mutant analysis showed that the glutamate residue corresponding to Asp(85) in bacteriorhodopsin acts as the primary acceptor of the Schiff-base proton in low-efficiency channelrhodopsins. Another photoactive-site residue corresponding to Asp(212) in bacteriorhodopsin serves as an alternative proton acceptor and plays a more important role in channel opening than the primary acceptor. In more efficient channelrhodopsins from Chlamydomonas reinhardtii, Mesostigma viride, and Platymonas (Tetraselmis) subcordiformis, the fast current was apparently absent. The inverse correlation of the outward proton transfer and channel activity is consistent with channel function evolving in channelrhodopsins at the expense of their capacity for active proton transport.
authors: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, TX, USA.
authors: Oleg A
authors: OA
authors: Sineshchekov
authors: Elena G
authors: EG
authors: Govorunova
authors: Jihong
authors: J
authors: Wang
authors: Hai
authors: H
authors: Li
authors: John L
authors: JL
authors: Spudich
mesh_terms: D001105:Archaea / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D000460:Chlorophyta / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D017136:Ion Transport; D008027:Light; D008564:Membrane Potentials / Q000235:genetics; D009154:Mutation; D011522:Protons; D012176:Retinoids / Q000737:chemistry / Q000378:metabolism; D025604:Rhodopsins, Microbial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D011522:Protons; D012176:Retinoids; D025604:Rhodopsins, Microbial; C121835:retinylidene chromophore; D001224:Aspartic Acid; D018698:Glutamic Acid