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SUMMARYThe KCR channelrhodopsins are recently-discovered light-gated ion channels with high K+selectivity, a property that has attracted broad attention among biologists– due to intense interest in creating novel inhibitory tools for optogenetics leveraging this K+selectivity, and due to the mystery of how this selectivity is achieved in the first place. Indeed, the molecular and structural mechanism for K+selectivity in KCRs has remained especially puzzling since these 7-transmembrane retinal-binding proteins completely lack structural similarity with known K+channels, which generally coordinate K+in a precisely symmetric conduction pathway formed by a tight interface among multiple small monomeric channel subunits (presumably not an accessible mechanism for the large KCR rhodopsin proteins). Here we present the cryo-electron microscopy structures of two KCRs fromHyphochytrium catenoideswith distinct spectral properties for light absorption and channel actuation,HcKCR1, andHcKCR2, at resolutions of 2.6 and 2.5 Å, respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal inHcKCR2, explaining the large spectral difference betweenHcKCR1 and 2. Next, our combined structural, electrophysiological, computational, and spectroscopic analyses revealed a new solution to the challenging problem of K+-selective transport. KCRs indeed do not exhibit the canonical tetrameric K+selectivity filter that specifically coordinates dehydrated K+; instead, single KCR monomers form a size exclusion filter using aromatic residues at the extracellular side of the pore which inhibits passage of bulky hydrated ions. This unique feature allows KCRs to function as K+channels under relevant physiological conditions, providing not only a novel mechanism for achieving high K+permeability ratios in biological ion channels, but also a framework for designing the next generation of inhibitory optogenetic tools.In BriefThe first structures of K+-selective channelrhodopsins (HcKCR1 and 2) are determined, revealing a K+selectivity mechanism distinctly different from canonical K+channels.HighlightsThe cryo-EM structures of K+-selective channelrhodopsins,HcKCR1 and 2, in nanodiscConditions under which naturally-occurring microbial rhodopsins have a 6-s-cisretinalIdentification of key residues for high K+permeability ratiosThe unique K+selectivity mechanism of KCRs

For efficient energy conversion and energy storage, developing highly efficient hydrogen permeable membranes is a crucial issue. Ceramic hydrogen permeable membranes, i.e., protonic electronic mixed conductors, work on bipolar transport of protons and electrons. Ceramic composite membranes meet this requirement with chemical and thermal stability, and they can be used not only as hydrogen permeable membranes but also as electrolytes in protonic ceramic fuel cells (PCFCs). Although improvement in protonic conductivity at heterointerfaces between constituting materials and/or space-charge relaxation around the resistive grain boundaries can be possible origins of the enhanced hydrogen permeability in the composite electrolyte membranes, its detailed mechanisms have not been clarified yet and the strategy of material design for composite membranes has not been established. In this study, the transport properties of composite electrolyte membranes consisting of protonic conductors and electronic conductors are investigated to realize highly efficient PCFC operating at low temperatures and to clarify the principles of carrier transport in the composite membranes. For the protonic conductor and the electronic conductor, we selected La2Ce2O7 (LDC) with a fluorite structure and La0.3Sr0.6TiO3 (LST) with a perovskite structure, respectively. The composite membrane composed of LDC and LST (LDC/LST) with various LDC to LST ratios was prepared by a solid-state reaction. Phase stability of LDC and LST were investigated by X-ray diffraction (XRD) measurements. Transport properties of hydrogen permeation cells using the LDC-LST composite membranes with Pt electrodes were assessed by electrochemical impedance spectroscopy (EIS) measurements and hydrogen permeation measurements. Hydrogen fluxes were measured by gas chromatography. The EIS measurements revealed that the grain-boundary resistance was reduced for the LDC/LST composite membrane in comparison with an LDC membrane. In particular, LDC/LST (weight ratio 1:2) showed the smallest grain-boundary resistance of 26 Ω cm at 600°C under 1%H2/Ar among the measured samples. Besides, the hydrogen permeation measurement showed that LDC/LST exhibited higher hydrogen permeability than the LDC membrane. In addition, the hydrogen flux of LDC/LST (1:2) was almost twice as high as that of LDC at 600°C. The low grain-boundary resistance and the high hydrogen permeability of LDC/LST composite membrane suggests that the introduction of LST accelerates the proton conduction in the membrane.

Middle rhodopsin (MR) found from the archaeon Haloquadratum walsbyi is evolutionarily located between two different types of rhodopsins, bacteriorhodopsin (BR) and sensory rhodopsin II (SRII). Some isomers of the chromophore retinal and the photochemical reaction of MR are markedly different from those of BR and SRII. In this study, to obtain the structural information regarding its active center (i.e., retinal), we subjected MR embedded in lipid bilayers to solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectroscopy. The analysis of the isotropic 13C chemical shifts of the retinal chromophore revealed the presence of three types of retinal configurations of dark-adapted MR: (13-trans, 15-anti (all-trans)), (13-cis, 15-syn), and 11-cis isomers. The higher field resonance of the 20-C methyl carbon in the all-trans retinal suggested that Trp182 in MR has an orientation that is different from that in other microbial rhodopsins, owing to the changes in steric hindrance associated with the 20-C methyl group in retinal. 13Cζ signals of Tyr185 in MR for all-trans and 13-cis, 15-syn isomers were discretely observed, representing the difference in the hydrogen bond strength of Tyr185. Further, 15N NMR analysis of the protonated Schiff base corresponding to the all-trans and 13-cis, 15-syn isomers in MR showed a strong electrostatic interaction with the counter ion. Therefore, the resulting structural information exhibited the property of stable retinal conformations of dark-adapted MR.

Optimization of Pt-based electrode structure is a key to enhance power generation performance of fuel cells and to reduce the Pt loading. This paper presents a new methodology for anode fabrication for solid acid fuel cells (SAFCs) operating at ca. 200 °C. Our membrane electrode assembly for SAFCs consisted of a CsH2PO4/SiP2O7 composite electrolyte and Pt-based electrodes. To obtain the anode, a commercial Pt/C catalyst and carbon substance, such as carbon black and carbon nanofiber, were mixed. The composite anode with Pt loading = 0.5 mg cm−2 demonstrated superior current-voltage characteristics to a benchmark Pt/C anode with Pt loading = 1 mg cm−2. We consider that the mixing of Pt/C catalyst and carbon substrate facilitated H2 mass transfer and increased the number of active sites.

The application of combined nitrogen, especially nitrate, to soybean plants is known to strongly inhibit nodule formation, growth and nitrogen fixation. In the present study, we measured the effects of supplying 5 mM nitrate on the growth of nodules, primary root, and lateral roots under light at 28 °C or dark at 18 °C conditions. Photographs of the nodulated roots were periodically taken by a digital camera at 1-h intervals, and the size of the nodules was measured with newly developed computer software. Nodule growth was depressed approximately 7 h after the addition of nitrate under light conditions. The nodule growth rate under dark conditions was almost half that under light conditions, and nodule growth was further suppressed by the addition of 5 mM nitrate. Similar results were observed for the extending growth rate of the primary root as those for nodule growth supplied with 5 mM nitrate under light/dark conditions. In contrast, the growth of lateral roots was promoted by the addition of 5 mM nitrate. The 2D-PAGE profiles of nodule protein showed similar patterns between the 0 and 5 mM nitrate treatments, which suggested that metabolic integrity may be maintained with the 5 mM nitrate treatment. Further studies are required to confirm whether light or temperature condition may give the primary effect on the growth of nodules and roots.