Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 137, Issue 9, Pages 3291-3299Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ja511788f
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- Japanese Ministry of Education, Culture, Sports, Science, and Technology [26708001, 26115706, 26620005, 23790060, 23590062, 26460044, 25104004, 25291034, 25104009, 23687019, 24121712]
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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 30100 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.
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