Biology subjects

Inoue, K.

Publications and source records attributed to Inoue, K..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Transcriptional Dynamics Uncover the Role of BNIP3 in Mitophagy during Muscle Remodeling in Drosophila

Differentiated muscle cells contain myofibrils and well-organized organelles, enabling powerful contractions. Muscle cell reorganization occurs in response to various physiological stimuli; however, the mechanisms behind this remodeling remain enigmatic due to the lack of a genetically trackable system. Previously, we reported that a subset of larval muscle cells is remodeled into adult abdominal muscle through an autophagy-dependent mechanism in Drosophila. To unveil the underlying mechanisms of this remodeling, we performed a comparative time-course RNA-seq analysis of isolated muscle cells with or without autophagy. It revealed both transcriptional dynamics independent of autophagy and highlighted the significance of BNIP3-mediated mitophagy in muscle remodeling. Mechanistically, we found that BNIP3 recruits autophagic machinery to mitochondria through its LC3-interacting (LIR) motif and minimal essential region (MER), which interact with Atg8a and Atg18a, respectively. Loss of BNIP3 leads to a substantial accumulation of larval mitochondria, ultimately impairing muscle remodeling. In summary, this study demonstrates that BNIP3-dependent mitophagy is critical for orchestrating the dynamic process of muscle remodeling. Impact statementA time-course RNA-seq analysis of muscle remodeling reveals transcriptional dynamics independent of autophagy and highlights the significance of BNIP3 in mitochondrial degradation in vivo.

cell biology↗

bioRxiv2024

Carotenoid pigments enhance rhodopsin-mediated phototrophy by light-harvesting and photocycle-accelerating

Microbial rhodopsins, photoreceptor proteins widely distributed in marine microorganisms, receive large amount of light energy that sustains marine ecosystems. Although rhodopsins generally harbor retinal as their only chromophore, a recent study reported that carotenoid antennae transfer light energy to the retinal in proteorhodopsins, proton pump rhodopsins abundant in marine environments. Here, using marine bacterial isolates, we detected energy transfer from a carotenoid (myxol) to retinal not only in proteorhodopsin but also in the chloride ion-pumping rhodopsin. Carotenoid binding improved the light utilization efficiency of the proteorhodopsin by accelerating the photocycle, together with facilitating light-harvesting. The carotenoid-binding ability is conserved in rhodopsins of the phylum Bacteroidota, which are widely transcribed in the photic zone. These findings suggest that the distribution of carotenoid-binding rhodopsins is more taxon-specific than previously thought, thus underscoring the importance of carotenoid-binding rhodopsins that provide an extended light utilization strategy in the environmental adaptation of marine bacteria.

microbiology↗

bioRxiv2024

Osmoregulation affects elimination of microplastics in fish in freshwater and marine environments

In recent decades, microplastics (MPs) have emerged as one of the biggest environmental challenges in aquatic environments. Ingestion and toxicity of MPs in seawater (SW) and freshwater (FW) fish have been studied extensively both in field and laboratory settings. However, the basic mechanism of how fish deal with MPs in SW and FW remains unclear, although physiological conditions of fish differ significantly in the two environments. In this study, using Javanese medaka (Oryzias javanicus), a euryhaline fish which adapts readily to both SW and FW, we investigated elimination of MPs in fish in SW and FW environments. We exposed O. javanicus larvae (21 days post-hatching) to 0.25 mg/L of fluorescent polystyrene microspheres (1 {micro}m) for 24 h and then conducted an elimination test for up to 5 days. The results showed that the gut retention time (GRT) of MPs is longer in FW than in SW, indicating that MP elimination occurs more quickly in SW than in FW. However, despite shorter GRTs, higher numbers of MPs tended to be retained for a longer period in SW larvae than FW larvae. Subsequently, using a fluorescent marker, gastrointestinal fluid was found to move more rapidly in the SW group. This finding indicates that water drinking accelerates gastrointestinal fluid movement, which moves MPs through the gut in SW larvae. Beside the difference in physiological conditions, MP elimination was faster when food was available, suggesting that feeding also affects MP elimination in fish. Internal factors such as body size and intestine length were also examined, but indicated no significant difference between SW and FW. Therefore, osmoregulation and feeding both influence MP elimination in fish.

physiology↗

bioRxiv2024

A coral peptide has bactericidal activity against a global marine pathogen, Vibrio coralliilyticus

Scleractinian corals and their associated microorganisms, including endosymbiotic dinoflagellates and bacteria, constitute coral holobionts. Rising seawater temperatures weaken coral symbiotic relationships, thereby increasing susceptibility to infectious diseases and leading to disease outbreaks and subsequent population declines. The temperature-dependent coral pathogen, Vibrio coralliilyticus, poses one of the greatest threats to coral reefs because of global warming. However, coral immune defenses against this pathogen are poorly understood. We previously identified coral genes responding to V. coralliilyticus exposure by transcriptomic analysis of the reef-building coral, Acropora digitifera. Here, bioinformatic analysis identified digitiferin, a coral antimicrobial peptide (AMP), in the genome of A. digitifera. Recombinant digitiferin showed antibacterial activity against both Gram-positive and -negative bacteria. A 3D structural model and direct microscopic observations revealed that digitiferin damaged bacterial cell membranes by forming pores; however, initial growth inhibition tests in 1.5 % (w/v) NaCl showed no effect on pathogens. Because we found that digitiferin is secreted from epithelial cells into mucus and shows salt sensitivity, we hypothesized that it is active against pathogens in low-salt environments. We then investigated whether it showed antibacterial activity against pathogens in low-salt, moderate-osmolality conditions, using mannitol for osmoregulation instead of NaCl. The results showed that digitiferin exhibits bactericidal activity against V. coralliilyticus under salt-free conditions. To the best of our knowledge, this is the first study to characterize a scleractinian AMP that kills V. coralliilyticus. These findings contribute to a better understanding of coral immunity and may facilitate development of techniques to overcome coral Vibrio disease. SignificanceHealthy coral reefs are one of the most important marine ecosystems, supporting [~]25 % of all marine organisms. Coral reef ecosystems are threatened by coral infectious diseases, as rising seawater temperatures exacerbate pathogen infectivity. Thus, understanding coral immune systems is becoming increasingly important to protect coral reefs. This study reports the discovery of an antimicrobial peptide, digitiferin, from the reef-building coral, Acropora digitifera. Digitiferin is effective against Gram-positive and -negative bacteria, including the major coral pathogen, Vibrio coralliilyticus, and is secreted into mucus from ectodermal granular epithelial cells. As the first antimicrobial peptide known to have bactericidal activity against V. coralliilyticus, this discovery enhances our understanding of coral immunology and coral-pathogen interactions.

microbiology↗

bioRxiv2024

Light-harvesting by antenna-containing rhodopsins in pelagic Asgard archaea

Aquatic bacterial rhodopsin proton pumps have been recently reported to utilize hydroxylated carotenoids1,2. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we report on light energy transfer from diverse hydroxylated carotenoids (lutein, diatoxanthin, and fucoxanthin) to heimdallarchaeial rhodopsins (HeimdallRs)3,4 from uncultured marine planktonic members of the "Ca. Kariarchaeaceae" ("Ca. Asgardarchaeota")5. These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light absorbing retinal chromophore within HeimdallRs. Furthermore, antenna enhancement of proton pumping by HeimdallRs is also observed under white-light illumination along with a carotenoid-binding-induced structural change in the protein. Our results indicate that the use of light-harvesting antennas in microbial rhodopsins is observed not only in bacteria but also in marine archaea.

microbiology↗

bioRxiv2024

Protonation/deprotonation-driven switch for the redox stability of low-potential ferredoxin

Ferredoxin is a small iron-sulfur protein and acts as an electron carrier. Low-potential ferredoxins harbor [4Fe-4S] cluster(s), which play(s) a crucial role as the redox center. Low-potential ferredoxins are able to cover a wide range of redox potentials (-700 to -200 mV); however, the mechanisms underlying the factors which control the redox potential are still enigmatic. Here, we determined the neutron structure of ferredoxin from Bacillus thermoproteolyticus, and experimentally revealed the exact hydrogen-bonding network involving the [4Fe-4S] cluster. The density functional theory calculations based on the hydrogen-bonding network revealed that protonation states of the sidechain of Asp64 close to the [4Fe-4S] cluster critically affected the stability of the reduced state in the cluster. These findings provide the first identification of the intrinsic control factor of redox potential for the [4Fe-4S] cluster in low-potential ferredoxins.

biochemistry↗

bioRxiv2024

Significance of extended N-terminal region and a first motif residue in a third transmembrane helix of a novel pump-like cation channelrhodopsin HulaCCR

Channelrhodopsins are light-gated ion channels consisting of seven-transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the "DTD motif" in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.

biophysics↗

bioRxiv2024

Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction

Chronic kidney disease (CKD) and atherosclerotic heart disease, frequently associated with dyslipidemia and hypertension, represent significant health concerns. We investigated the interplay among these conditions, focusing on the role of oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) in renal injury via G protein q subunit (Gq) signaling. We hypothesized that oxLDL enhances Ang II-induced Gq signaling via the AT1 (Ang II type 1 receptor)-LOX1 (lectin-like oxLDL receptor) complex. Based on CHO and renal cell model experiments, oxLDL alone did not activate Gq signaling. However, when combined with Ang II, it significantly potentiated Gq-mediated inositol phosphate 1 production and calcium influx in cells expressing both LOX-1 and AT1 but not in AT1-expressing cells. This suggests a critical synergistic interaction between oxLDL and Ang II in the AT1-LOX1 complex. Conformational studies using AT1 biosensors have indicated a unique receptor conformational change due to the oxLDL-Ang II combination. In vivo, wild-type mice fed a high-fat diet with Ang II infusion presented exacerbated renal dysfunction, whereas LOX-1 knockout mice did not, underscoring the pathophysiological relevance of the AT1-LOX1 interaction in renal damage. These findings highlight a novel mechanism of renal dysfunction in CKD driven by dyslipidemia and hypertension and suggest the therapeutic potential of AT1-LOX1 receptor complex in patients with these comorbidities.

molecular biology↗

bioRxiv2024

SLC35G1: A highly chloride-sensitive transporter responsible for the basolateral membrane transport in intestinal citrate absorption

The intestinal absorption of essential nutrients, especially those not readily biosynthesized, is a critical physiological process for maintaining homeostasis. Numerous studies have indicated that intestinal absorption is mediated by various membrane transporters. Citrate, a crucial bioactive compound produced as an intermediate in the Krebs cycle, is absorbed in the small intestine through carrier-mediated systems because of its high hydrophilicity. While the luminal absorption of citrate is mediated by Na+-dicarboxylate cotransporter 1 (NaDC1/SLC13A2), the mechanism governing the release of the transported citrate into the bloodstream remains unknown. Here, we explored the transporters responsible for intestinal citrate absorption at the basolateral membrane, focusing on highly expressed orphan transporters in the small intestine as candidates. Consequently, SLC35G1, originally identified as a partner of stromal interaction molecule 1, a cell surface transmembrane glycoprotein, was found to play a role in the intestinal absorption of citrate at the basolateral membrane. Furthermore, our results revealed that SLC35G1-mediated citrate transport was diminished by chloride ions at physiologically relevant extracellular concentrations. This suggests that SLC35G1, to our best knowledge, is the first transporter identified to be extremely sensitive to chloride ions among those functioning on the basolateral membrane of intestinal epithelial cells. This study provides valuable insights into the intestinal absorption of citrate and significantly contributes to elucidating the poorly understood molecular basis of the intestinal absorption system.

physiology↗

bioRxiv2024

Element accumulation in the tracheal and bronchial cartilages of monkeys

Compositional changes in the tracheal and bronchial cartilages can affect respiratory ventilation and lung function. We aimed to elucidate element accumulation in the tracheal and bronchial cartilages of monkeys and divided it into four sites: tracheal, tracheal bifurcation, left bronchial, and right bronchial cartilages. The elemental content was analyzed using inductively coupled plasma atomic emission spectrometry. The average calcium content was two to three times higher in the tracheal cartilage than in the other three cartilages. The trends of phosphorus and zinc were similar to those of calcium. The average calcium, phosphorus, and zinc contents were the highest in the tracheal cartilage and decreased in the following order: the left bronchial, right bronchial, and tracheal bifurcation cartilages. These findings revealed that differences existed in element accumulation between different sites within the same airway cartilage and that calcium, phosphorus, and zinc accumulation mainly occurred in the tracheal cartilage. A substantial direct correlation was observed between age and calcium content in the tracheal and bronchial cartilages and all such monkeys with high calcium content were > four years of age. These results suggest that calcium accumulation occurs in the tracheal and bronchial cartilages after reaching a certain age. An extremely substantial direct correlation was observed between calcium and phosphorus contents in the tracheal and bronchial cartilages. This finding is similar to the previously published calcium and phosphorus correlations in several other cartilages, suggesting that the calcium and phosphorus contents of cartilage exist in a certain ratio.

zoology↗

bioRxiv2023

A single-cell atlas of transcribed cis-regulatory elements in the human genome

Transcribed cis-regulatory elements (tCREs), such as promoters and enhancers, are fundamental to modulate gene expression and define cell identity. The detailed mapping of tCREs at single-cell resolution is essential for understanding the regulatory mechanisms that govern cellular functions. Prior tCRE catalogs, limited by bulk analysis, have often overlooked cellular heterogeneity. We have constructed a tCRE atlas using single-cell 5-RNA-seq, capturing over 340,000 single-cells from 23 human tissues and annotating more than 175,000 tCREs, substantially enhancing the scope and granularity of existing cis-regulatory element annotations in the human genome. This atlas unveils patterns of gene regulation, revealing connections between broadly expressed promoters and cell type-specific distal tCREs. Assessing trait heritability at single-cell resolution with a novel tCRE module-based approach, we uncovered the nuanced trait-gene regulatory relationships across a continuum of cell populations, offering insights beyond traditional gene-level and bulk-sample analyses. Our study bridges the gap between gene regulation and trait heritability, underscoring the potential of single-cell analysis to elucidate the genetic foundations of complex traits. These insights set the stage for future research to investigate the impact of genetic variations on diseases at the individual level, advancing the understanding of cellular and molecular basis of trait heritability.

genomics↗

bioRxiv2023

Blocking Formation of Neurotoxic Reactive Astrocytes is Beneficial Following Stroke

Microglia and astrocytes play an important role in the neuroinflammatory response and contribute to both the destruction of neighboring tissue as well as the resolution of inflammation following stroke. These reactive glial cells are highly heterogeneous at both the transcriptomic and functional level. Depending upon the stimulus, microglia and astrocytes mount a complex, and specific response composed of distinct microglial and astrocyte substates. These substates ultimately drive the landscape of the initiation and recovery from the adverse stimulus. In one state, inflammation- and damage-induced microglia release tumor necrosis factor (TNF), interleukin 1 (IL1), and complement component 1q (C1q), together TIC. This cocktail of cytokines drives astrocytes into a neurotoxic reactive astrocyte (nRA) substate. This nRA substate is associated with loss of many physiological astrocyte functions (e.g., synapse formation and maturation, phagocytosis, among others), as well as a gain-of-function release of neurotoxic long-chain fatty acids which kill neighboring cells. Here we report that transgenic removal of TIC led to reduction of gliosis, infarct expansion, and worsened functional deficits in the acute and delayed stages following stroke. Our results suggest that TIC cytokines, and likely nRAs play an important role that may maintain neuroinflammation and inhibit functional motor recovery after ischemic stroke. This is the first report that this paradigm is relevant in stroke and that therapies against nRAs may be a novel means to treat patients. Since nRAs are evolutionarily conserved from rodents to humans and present in multiple neurodegenerative diseases and injuries, further identification of mechanistic role of nRAs will lead to a better understanding of the neuroinflammatory response and the development of new therapies.

neuroscience↗

bioRxiv2023

mRNA vaccine induces protective immunity against the type III secretory virulence of Pseudomonas aeruginosa

Few studies have addressed mRNA vaccination against bacteria despite the desperate need to control antimicrobial resistance. Herein, we developed mRNA vaccines targeting the type III secretion system of Pseudomonas aeruginosa, which plays a critical role in the pathogenesis. Vaccination with a nucleoside-modified mRNA encapsulated in lipid nanoparticles improved mouse survival, reduced the bacterial load in the lung, and alleviated pathological changes in the lungs after bacterial challenge.

immunology↗

bioRxiv2023

Biosynthesis of gibberellin-related compounds modulates far-red light responses in the liverwort Marchantia polymorpha

The phytohormone gibberellins (GAs) are key regulators of growth, development and environmental responses in angiosperms. From an evolutionary perspective, all major steps of GA biosynthesis are conserved among vascular plants, while GA biosynthetic intermediates such as ent-kaurenoic acid (KA) are also produced by bryophytes. Here we show that in the liverwort Marchantia polymorpha, KA and GA12 are synthesized by evolutionarily conserved enzymes, which are required for developmental responses to far-red light (FR). Under FR-enriched conditions, mutants of various biosynthesis enzymes consistently altered thallus growth allometry, delayed the initiation of gametogenesis, and affected the morphology of gamete-bearing structures (gametangiophores). By chemical treatments and LC-MS/MS analyses, we confirmed these phenotypes were caused by deficiency of some GA-related compounds derived from KA, but not bioactive GAs from vascular plants. Transcriptome analysis showed that FR enrichment induced the up-regulation of genes related to stress responses and secondary metabolism in M. polymorpha, which was largely dependent on the biosynthesis of GA-related compounds. Due to the lack of the canonical GA receptors in bryophytes, we hypothesize that GA-related compounds are commonly synthesized in land plants but co-opted independently to regulate responses to light quality change in different lineages during the past 450 million years of evolution.

plant biology↗

bioRxiv2023

The defects of the hippocampal ripples and theta rhythm in depression, and the effects of physical exercise on their amelioration.

Accumulated evidence demonstrate that environmental stress affects the hippocampus, functioning in cognition and sociality, and causes various depressive symptoms. In addition, recent findings showed that environmental stress influenced the hippocampal activity correlated with neuroinflammation, and impaired the hippocampal sharp wave ripples (SWRs), pattens of spike sequences, and the theta rhythms, a strong oscillation observed in the hippocampus. The involvement of the electrophysiological alterations in the etiology of depression has not been appreciated especially in the hippocampus. Furthermore, the pathological markers associated with such alterations have not been identified. In the present study, therefore, the impairment of the SWRs and the theta rhythms in the hippocampus of the restraint stress-induced depression model of mice was analyzed. In the model mice the hippocampal SWRs and theta rhythms were impaired in depression, while physical exercise significantly reverted them. As previously reported, chronic stress induced inflammation in the affected hippocampus in parallel with defects of adult neurogenesis, on the other hand physical exercise ameliorated those pathological conditions of the bran in depression. In conclusion, this study demonstrated the implications of impairment of the hippocampal SWRs and theta rhythms in the etiology of depression and their usefulness as diagnostic markers of depression.

neuroscience↗

bioRxiv2023

Reprogramming Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD1 Therapy

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Anti-PD-L1 immunotherapy combined with gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers, but responses are seen only in a minority of patients. Here, we studied the roles of anti-PD1 and anti-CTLA-4 immune checkpoint blockade (ICB) therapies when combined with gemcitabine/cisplatin and the mechanisms of treatment benefit in orthotopic murine ICC models. We evaluated the effects of the combined treatments on ICC vasculature and immune microenvironment using flow cytometry analysis, immunofluorescence, imaging mass cytometry, RNA-sequencing, qPCR, and in vivo T-cell depletion and CD8+ T-cell transfer using orthotopic ICC models and transgenic mice. Combining gemcitabine/cisplatin with anti-PD1 and anti-CTLA-4 antibodies led to substantial survival benefits and reduction of morbidity in two aggressive ICC models, which were ICB-resistant. Gemcitabine/cisplatin treatment increased the frequency of tumor-infiltrating lymphocytes and normalized the ICC vessels, and when combined with dual CTLA-4/PD1 blockade, increased the number of activated CD8+Cxcr3+IFN-{gamma}+ T-cells. Depletion of CD8+ but not CD4+ T-cells compromised efficacy. Conversely, CD8+ T-cell transfer from Cxcr3-/- versus Cxcr3+/+ mice into Rag1-/- immunodeficient mice restored the anti-tumor effect of gemcitabine/cisplatin/ICB combination therapy. Finally, rational scheduling of the ICBs (anti-CTLA-4 "priming") with chemotherapy and anti-PD1 therapy achieved equivalent efficacy with continuous dosing while reducing overall drug exposure. In summary, gemcitabine/cisplatin chemotherapy normalizes vessel structure, increases activated T-cell infiltration, and enhances anti-PD1/CTLA-4 immunotherapy efficacy in aggressive murine ICC. This combination approach should be clinically tested to overcome resistance to current therapies in ICC patients. One Sentence SummaryImmune microenvironment reprogramming by chemotherapy and priming using CTLA-4 blockade render ICCs responsive to anti-PD-1 immunotherapy.

cancer biology↗

bioRxiv2022

Transcriptional lability of brain oxytocin receptor (Oxtr) generates diversity in brain OXTR distribution and social behaviors

Although oxytocin (OXT) exhibits a highly conserved neuroanatomical pattern among vertebrates, the distribution of OXT receptor (OXTR) in brain varies considerably across species and is associated with species-typical social behavior. To investigate the genomic basis of the phylogenetic plasticity in brain Oxtr expression and its social behavioral consequences, we generated transgenic mice carrying a bacteria artificial chromosome (BAC) harboring the entire prairie vole Oxtr locus and flanking intergenic regulatory regions. We established eight independent "volized" mouse lines expressing prairie vole Oxtr (pvOxtr). Strikingly, despite conserved Oxtr expression in mammary gland of all transgenic mouse lines, each line displayed a unique pattern of brain expression distinct from both mice and prairie voles. Together with topologically associating domain (TAD) structure analysis with mouse genome, our findings suggest that unlike Oxt, Oxtr expression patterns in brain, involve contributions of distal regulatory elements beyond the BAC insert. In contrast, Oxtr expression in peripheral tissues appears resistant to such distal influences. Moreover, the "volized" mouse lines with different brain Oxtr expression patterns showed differences in partner preference and maternal behaviors, providing direct functional evidence that variation in brain Oxtr expression can drive differences in social behaviors. We propose that brain Oxtr expression is transcriptionally sensitive to long-range interactions with distal genomic elements, rendering it more susceptible to diverse regulatory influences. This supports a model in which regulatory flexibility facilitates the evolutionary diversification of social behavior, while maintaining essential peripheral Oxtr expression.

neuroscience↗

bioRxiv2022

Structural basis for ion selectivity in potassium-selective channelrhodopsins

The 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 from Hyphochytrium catenoides with distinct spectral properties for light absorption and channel actuation, HcKCR1, and HcKCR2, at resolutions of 2.6 and 2.5 [A], respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal in HcKCR2, explaining the large spectral difference between HcKCR1 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. HighlightsO_LIThe cryo-EM structures of K+-selective channelrhodopsins, HcKCR1 and 2, in nanodisc C_LIO_LIConditions under which naturally-occurring microbial rhodopsins have a 6-s-cis retinal C_LIO_LIIdentification of key residues for high K+ permeability ratios C_LIO_LIThe unique K+ selectivity mechanism of KCRs C_LI

biophysics↗