title: Converting a light-driven proton pump into a light-gated proton channel.
abstract: There are two types of membrane-embedded ion transport machineries in nature. The ion pumps generate electrochemical potential by energy-coupled active ion transportation, while the ion channels produce action potential by stimulus-dependent passive ion transportation. About 80% of the amino acid residues of the light-driven proton pump archaerhodopsin-3 (AR3) and the light-gated cation channel channelrhodopsin (ChR) differ although they share the close similarity in architecture. Therefore, the question arises: How can these proteins function differently? The absorption maxima of ion pumps are red-shifted about 30-100 nm compared with ChRs, implying a structural difference in the retinal binding cavity. To modify the cavity, a blue-shifted AR3 named AR3-T was produced by replacing three residues located around the retinal (i.e., M128A, G132V, and A225T). AR3-T showed an inward H(+) flux across the membrane, raising the possibility that it works as an inward H(+) pump or an H(+) channel. Electrophysiological experiments showed that the reverse membrane potential was nearly zero, indicating light-gated ion channeling activity of AR3-T. Spectroscopic characterization of AR3-T revealed similar photochemical properties to some of ChRs, including an all-trans retinal configuration, a strong hydrogen bond between the protonated retinal Schiff base and its counterion, and a slow photocycle. From these results, we concluded that the functional determinant in the H(+) transporters is localized at the center of the membrane-spanning domain, but not in the cytoplasmic and extracellular domains.
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Inoue
authors.forename: Takashi
authors.initials: T
authors.lastname: Tsukamoto
authors.forename: Kazumi
authors.initials: K
authors.lastname: Shimono
authors.forename: Yuto
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Seiji
authors.initials: S
authors.lastname: Miyauchi
authors.forename: Shigehiko
authors.initials: S
authors.lastname: Hayashi
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sudo
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005260:Female; D025602:Halorhodopsins / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D008027:Light; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D025604:Rhodopsins, Microbial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025603:Sensory Rhodopsins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: D025602:Halorhodopsins; D017494:Proton Pumps; D011522:Protons; D011994:Recombinant Proteins; D025604:Rhodopsins, Microbial; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; D012243:Rhodopsin; D012172:Retinaldehyde