Biology subjects

Kawano, T.

Publications and source records attributed to Kawano, T..

16 recordsLinked to original sources

bioRxiv2026

GAT1508, a selective neuronal GIRK1/2 activator, exhibits superior antiepileptic efficacy and synergizes with cannabidiol

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels are key regulators of neuronal excitability and represent promising therapeutic targets for epilepsy. Here, we characterize GAT1508, a urea-based small-molecule activator selectively targeting neuronal GIRK1/2 heterotetramers, and compare its efficacy with previously described urea compounds ML297 and GiGA1. In a pentylenetetrazole (PTZ)-induced seizure model, GAT1508 demonstrated robust antiseizure activity, outperforming both ML297 and GiGA1 and achieving efficacy comparable to valproic acid. Electrophysiological studies using two-electrode voltage clamp in Xenopus oocytes revealed that GAT1508, unlike ML297 and GiGA1, selectively activates GIRK1/2 channels without significant activation of cardiac GIRK1/4 channels, suggesting reduced risk of cardiovascular side effects. Structural and computational analyses indicate that enhanced stabilization of GAT1508 compared to ML297 within the GIRK1/2 binding pocket promotes both ion permeation and channel gating through coordinated interactions with key residues. Furthermore, GAT1508, like the two other urea drugs, exhibited synergistic activation of GIRK channels when combined with G{beta}{gamma}-mediated pathways and significantly potentiated the antiseizure effects of cannabidiol (CBD) in both hippocampal slice recordings and in vivo seizure models. These findings establish GAT1508 as a potent and selective GIRK1/2 activator and support a novel combination therapeutic strategy for epilepsy based on convergent GIRK channel modulation.

pharmacology and toxicology↗

bioRxiv2026

Asymmetric assembly underlies activation mechanism of neuronal GIRK1/2 channels

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels are principal effectors of inhibitory GPCR signaling. Here we combine cryo-electron microscopy, single-molecule imaging, electrophysiology and molecular dynamics to define the architecture and activation of neuronal GIRK1/2 channels. GIRK1/2 preferentially assembles as an asymmetric heterotetramer containing one GIRK1 and three GIRK2 subunits. Three PIP2; molecules bound to GIRK2 promote partial cytoplasmic-domain docking, whereas engagement of a fourth PIP2 by GIRK1 completes docking and opens the helix bundle crossing gate. GIRK1 also remodels the electrostatic environment beneath the selectivity filter, where its E141 and D173 residues create a permeation module that enhances K+ conduction. The GIRK1-selective activator ML297 binds at the GIRK1-GIRK2 interface and controls both permeation and gating. These findings establish how asymmetric assembly couples sequential lipid-dependent gating to efficient K+ permeation and GIRK1-dependent pharmacological activation.

biophysics↗

bioRxiv2026

Stoichiometry-dependent gating and structural pharmacology of cardiac GIRK1/4 channels

Cardiac G protein-gated inwardly rectifying potassium-K+ (GIRK) channels, formed by GIRK1/4 heterotetramers or GIRK4 homotetramers, are crucial regulators of heart rate, and their dysfunction is associated with atrial fibrillation. Despite their therapeutic potential as anti-arrhythmic targets, development of selective GIRK modulators has been limited by an incomplete understanding of their structures, gating mechanisms, and pharmacology. Here, we report high-resolution structures of the GIRK4 homotetramer and of GIRK1/4 heterotetramers, revealing that they form in unexpected 1:3 and 2:2 assembly stoichiometries that we corroborate using cell-based single molecule microscopy. Electrophysiological assays and molecular dynamics simulations further show that incorporation of one or two GIRK1 subunits into GIRK4-containing channels progressively opens the helix bundle crossing (HBC) gate and increases basal channel activity. Additional structures of GIRK1/4 complexes with ML297, a small-molecule ligand selective for GIRK1-containing channels, reveal distinct GIRK1-centered intersubunit binding environments. Remarkably, ML297 inhibits GIRK1/4 channels with a 1:3 stoichiometry but potentiates channels with a 2:2 stoichiometry, establishing a direct link between subunit composition, ligand-binding environment, and pharmacological efficacy. Together, these findings provide a structural and mechanistic framework for the rational design of ligands with enhanced selectivity among cardiac and neuronal GIRK channel heterotetramers.

molecular biology↗

bioRxiv2026

Zuranolone mitigates delirium-like bispectral EEG changes, behavioral deficits, and neuroinflammation across surgical and inflammatory mouse models and age groups

Delirium is an acute, fluctuating brain dysfunction that commonly follows surgery and systemic inflammation, disproportionately affects older adults, and remains difficult to quantify continuously over time and treat pharmacologically. Here, we tested whether the neuroactive steroid zuranolone, a positive allosteric modulator of synaptic and extrasynaptic GABA_A receptors, mitigates delirium-like abnormalities across two complementary murine delirium models, a lipopolysaccharide-induced systemic inflammation (LPS) model and a postoperative delirium (POD) model, primarily in young and aged mice, with selected analyses in super-aged mice. Using continuous EEG with a validated bispectral EEG (BSEEG) metric, we found that zuranolone attenuated delirium-like EEG slowing in the LPS model in young mice in a dose-dependent manner and retained efficacy in aged mice. In the POD model, prophylactic dosing provided limited benefit in young mice, whereas post-surgery treatment reduced postoperative BSEEG elevations. In aged mice, prophylactic dosing suppressed POD-associated BSEEG abnormalities, and in super-aged mice, prophylactic zuranolone improved survival after POD induction. In parallel, zuranolone reduced microglial density and activation markers (IBA1 and CD68 immunoreactivity) at 24 h after POD surgery and after LPS challenge, with effects that were particularly evident in peri-screw site tissue in young POD mice and more broadly distributed across regions in aged mice. Finally, in young mice, zuranolone improved a composite behavioral severity score in the LPS model, whereas behavioral effects in the POD model were modest and domain-specific. Together, these findings support zuranolone as a candidate strategy to reduce delirium-like electrophysiological and neuroimmune abnormalities, with the strongest effects in inflammation-driven and age-vulnerable contexts.

neuroscience↗

bioRxiv2026

Distinct roles of directional and positional experience in de novo visuomotor learning

Humans can flexibly acquire entirely new sensorimotor mappings, a process known as de novo motor learning. A central challenge in de novo motor learning is that the learner must discover a viable solution from scratch within a highly redundant control space, without predefined task constraints. Understanding what types of sensorimotor information contribute to the formation of accurate motor behavior in such situations is therefore critical for explaining how novel sensorimotor skills are acquired. While previous studies have suggested that novel visuomotor mappings can be formed based on movement direction and target position, it remains unclear how these two types of information contribute to the learning process. To address this question, we trained 25 human participants to learn arbitrary joystick-to-cursor mapping. We then employed a generalization paradigm to selectively restrict learning experience to either movement direction or target position. Three distinct target conditions were designed: one emphasized target position (P), another emphasized movement direction (D), and a third (P&D) encouraged learning of both components separately. As a result, direction experience improved movement initiation, whereas position experience enhanced movement termination. However, in the P&D condition, combining these experiences did not yield additive generalization. Instead, endpoint accuracy was positively correlated with the degree of alignment between direction- and position-based joystick outputs within the control space. These results suggest that accurate formation of a novel sensorimotor map depends on the coordinated use of directional and positional experiences. Significant StatementHow do humans build entirely new sensorimotor relationships from scratch? This study examined how distinct sensorimotor experiences (movement direction and target position) contribute to the acquisition of a novel joystick-to-cursor mapping. By isolating these experiences, we found that direction experience improved movement initiation, while position experience enhanced movement termination. However, combining these experiences did not lead to more accurate movements as a whole. Instead, the accuracy was related to how well directional and positional joystick outputs were aligned in a control space. These findings suggest that de novo motor learning requires the coordinated use of directional and positional information.

neuroscience↗

bioRxiv2025

A novel transformer-based platform for the prediction and design of biosynthetic gene clusters for (un)natural products

Biosynthetic gene clusters (BGCs), comprising sets of functionally related genes responsible for synthesizing complex natural products, are a rich source of bioactive compounds with pharmaceutical potential. Here, we present a transformer-based framework that models functional domains as linguistic units to capture and predict their positional relationships within genomes. Using a RoBERTa architecture, we trained models on four progressively broader datasets: bacterial BGCs, Actinomycetes genomes, bacterial genomes, and bacterial plus fungal genomes. Evaluation using 2,492 experimentally-validated BGCs from the MIBiG database showed that more than 60% of true domains were ranked first and over 80% within the top 10 candidates. Our models also achieved classification accuracies exceeding 70% for major compound classes including polyketides (PKs) and terpenes. To explore model-guided BGC design, we compared predictions from the BGC-trained and genome-trained models using the BGC for the bacterial diterpenoid cyclooctatin as a case study. The genome-trained model uniquely predicted several domains absent from both the original BGC and the prediction by the BGC-trained model. Heterologous expression of one of those predicted domains in Streptomyces albus, together with the biosynthetic genes for cyclooctatin, yielded an unknown cyclooctatin derivative. This framework not only provides a novel BGC prediction method using machine learning but also facilitates rational design of artificial BGCs. Future integration of transcriptomic, protein structural, and phylogenetic data will enhance the models predictive and generative capabilities, supporting accelerated discovery and engineering of natural products. Author SummaryBGCs encode diverse natural products, including antibiotics and anticancer agents. Identifying and designing BGCs in microbial genomes is crucial for discovering new bioactive compounds. In this study, we developed a transformer-based deep learning model that treats protein domains as language-like tokens and learns how they are arranged in genomes. By training on both known BGCs and whole genomes, the model successfully predicts biologically plausible combinations of domains, including those absent in known BGCs. We experimentally validated one such prediction by expressing a newly identified gene alongside known cyclooctatin biosynthetic genes, confirming the production of an unknown cyclooctatin derivative. Our results demonstrate how language models can uncover hidden biosynthetic potential and offer a promising new AI tool for natural product discovery and synthetic biology.

bioinformatics↗

bioRxiv2025

Hypoxia Exacerbates Kir2.1 Channel Dysfunction in an Andersen-Tawil Syndrome Variant Through a SUMO-Dependent Mechanism

BackgroundAndersen-Tawil Syndrome type 1 (ATS1) is a multisystem channelopathy that predisposes patients to ventricular dysrhythmias and increases the risk of sudden cardiac death. ATS1 arises from loss-of-function mutations in Kir2.1, the inward rectifying potassium channel responsible for most of IK1 in ventricular cardiomyocytes. IK1 is suppressed by SUMOylation, a post- translational modification upregulated in hypoxia, a known proarrhythmic stimulus. We investigated whether current from the ATS1-linked variant Kir2.1-R67Q is inhibited by hypoxia and whether this suppression can be reversed by pharmacological inhibition of the SUMO pathway. MethodsWe used patch-clamp recording to measure IK1 and Kir2.1 currents under acute hypoxia, with and without the SUMO pathway inhibitor TAK-981. To quantify SUMOylation stoichiometry, we applied single molecule photobleaching. A multidisciplinary approach combining electrophysiology, molecular modeling, and optogenetic phosphoinositide was used to measure the impact of Kir2.1- R67Q and SUMOylation on channel interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), a required gating cofactor. ResultsKir2.1 can be modified by up to two SUMO proteins attached to diagonally opposite subunits, with each SUMOylation event reducing current by [~]20%. Heterozygouse channels containing two R67Q subunits were more susceptible to hypoxic suppression than wild type. TAK-981 blocked hypoxic inhibition of IK1 in ventricular cardiomyocytes and abolished Kir2.1 SUMOylation. In cells expressing Kir2.1-R67Q, TAK-981 significantly increased currents and mitigated hypoxic suppression. Computational modeling and optogenetic dephosphorylation revealed that both the R67Q mutation and converge to disrupt Kir2.1- PIP2 interactions, producing synergistic inhibition of channel function. ConclusionsHypoxia-induced SUMOylation and the R67Q mutation synergistically suppress Kir2.1 activity by impairing channel-PIP2 interactions. TAK-981 restores IK1 by preventing SUMOylation under hypoxic conditions and enhancing current through Kir2.1-R67Q channels. These findings support a two-hit model of arrhythmogenesis in ATS1 and identify SUMO pathway inhibition as a potential therapeutic strategy to reduce arrhythmic risk in affected patients.

physiology↗

bioRxiv2025

Orthosteric interactions with PIP2 activate TMEM16A channels

TMEM16A channels pass Ca2+-activated Cl- currents that drive a plethora of fundamental physiological processes. TMEM16A channels are activated by a rise in intracellular Ca2+ levels but also require interactions with the signaling phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to gate open. Although PIP2 is essential for the activity of many types of ion channels, its precise binding site and role in channel gating remain poorly understood in most cases, limiting efforts to study channel dynamics and design targeted modulators. In this study, we identify the PIP2 binding interactions that govern TMEM16A gating and permeation. Using a combination of gating molecular dynamics (GMD) simulations and electrophysiological assays, we reveal how the 4 helix of TMEM16A interacts with both the phosphate headgroups and acyl chains of PIP2 to open an electrostatic ring within the channel and stabilize the extracellular opening of the Cl- conduction pathway. These findings provide key insights into the dynamic role of PIP2 in membrane protein function and shed light on the activation mechanism of TMEM16A. This work establishes a framework for rational targeting of TMEM16A in drug development, with potential therapeutic applications in a variety of diseases.

biophysics↗

bioRxiv2024

Closed-loop electrical stimulation to prevent focal epilepsy progression and long-term memory impairment

Interictal epileptiform discharges (IEDs) are ubiquitously expressed in epileptic networks and disrupt cognitive functions. It is unclear whether addressing IED-induced dysfunction could improve epilepsy outcomes as most therapeutics target seizures. We show in a model of progressive hippocampal epilepsy that IEDs produce pathological oscillatory coupling which is associated with prolonged, hypersynchronous neural spiking in synaptically connected cortex and expands the brain territory capable of generating IEDs. A similar relationship between IED-mediated oscillatory coupling and temporal organization of IEDs across brain regions was identified in human subjects with refractory focal epilepsy. Spatiotemporally targeted closed-loop electrical stimulation triggered on hippocampal IED occurrence eliminated the abnormal cortical activity patterns, preventing spread of the epileptic network and ameliorating long-term spatial memory deficits in rodents. These findings suggest that stimulation-based network interventions that normalize interictal dynamics may be an effective treatment of epilepsy and its comorbidities, with a low barrier to clinical translation. One-Sentence SummaryTargeted closed-loop electrical stimulation prevents spread of the epileptic network and ameliorates long-term spatial memory deficits.

neuroscience↗

bioRxiv2023

Differentiation of Large Extracellular Vesicles in Oral Fluid: Combined Protocol of Small Force Centrifugation and Pattern Analysis

Extracellular vesicles (EVs) in biofluids are highly heterogeneous entities in terms of their origins and physicochemical properties. Considering the application of EVs in diagnostic and therapeutic fields, it is of extreme importance to establish differentiating methods by which focused EV subclasses are operationally defined. Several differentiation protocols have been proposed; however, they have mainly focused on smaller types of EVs, and the heterogeneous nature of large EVs has not yet been fully explored. In this report, to classify large EVs into subgroups based on their physicochemical properties, we have developed a protocol, named EV differentiation by sedimentation patterns (ESP), in which entities in the crude large EV fraction are first moved through a density gradient of iodixanol with small centrifugation forces, and then the migration patterns of molecules through the gradients are analyzed using a non-hierarchical data clustering algorithm. Based on this method, proteins in the large EV fractions of oral fluids clustered into three groups: proteins shared with small EV cargos and enriched in immuno-related proteins (Group 1), proteins involved in energy metabolism and protein synthesis (Group 2), and proteins required for vesicle trafficking (Group 3). These observations indicate that the physiochemical properties of EVs, which are defined through low-speed gradient centrifugation, are well associated with their functions within cells. This protocol enables the detailed subclassification of EV populations that are difficult to differentiate using conventional separation methods.

molecular biology↗

bioRxiv2021

Histidine-rich protein 2: a new pathogenic factor of Plasmodium falciparum malaria

Plasmodium falciparum causes serious malaria symptoms; when this protozoan parasite infects human erythrocytes, it produces and secretes large amounts of histidine-rich protein 2 (PfHRP2) into human blood. Thus, PfHRP2 is a well-known diagnostic marker for malaria infection. Here, however, we also identified PfHRP2 as a pathogenic factor produced by P. falciparum. PfHRP2 showed cell penetration and cytotoxicity against various human cells. In particular, PfHRP2 showed significant cytotoxicity over 5 days at the same concentration as in P. falciparum-infected patients blood (90-100 nM). This result is consistent with the mortality rate of P. falciparum malaria, which increases rapidly in untreated cases for 3-7 days. In addition, the cell penetration and cytotoxicity of PfHRP2 increased 2.5- and 2.6-fold, respectively, in the absence of serum, which suggests that low serum protein concentrations (occurring during malnutrition, for example) increase the risk of adverse effects from PfHRP2 (consistent with malnutrition increasing the lethality of malaria infection). We also showed that PfHRP2 bound to Ca2+ ions, localized to intracellular lysosomes, increased lysosomal Ca2+ levels, and inhibited the basal level of autophagy by inhibiting autolysosome formation. Furthermore, the Ca2+-dependent cytotoxicity of PfHRP2 was suppressed by the metal ion chelator ethylenediaminetetraacetic acid (EDTA). In summary, our findings suggest that PfHRP2 acts as a pathogenic factor in P. falciparum-infected patients and is associated with the exacerbation of malaria. Furthermore, EDTA is a promising candidate as a therapeutic agent for the suppression of PfHRP2 pathogenicity. Overall, this study provides new insights into P. falciparum malaria pathogenesis and treatment.

biochemistry↗

bioRxiv2021

Nuclear activation in dual-durotaxing cells on a matrix with cell-scale stiffness-heterogeneity

Living organisms are typically composed of various tissues with microscopic cell-scale stiffness-heterogeneity, in which some cells receive dynamically fluctuating mechanical stimuli from the heterogeneous extracellular milieu during long-term movement. Although intracellular stress dynamics (ISD), which are closely related to the regulation of cell functions such as proliferation and differentiation, can be characteristically modulated in cells migrating on a matrix with stiffness-heterogeneity, it has been unclear how the mode of fluctuation of ISD affects cell functions. In the present study, we demonstrate that mesenchymal stem cells (MSCs) dual-durotaxing (i.e., both forward and reverse durotaxis) on microelastically-patterned gels with stiff triangular domains markedly amplify the fluctuation of ISD, nuclear shape, and the spatial distribution of chromatins, which makes the cells remain far from tensional equilibrium. We provide evidence that amplified chromatin fluctuation in the dual-durotaxing MSCs can cause activation of cellular vigor and maintenance of the stemness.

biophysics↗

bioRxiv2020

Diagnosis of central serous chorioretinopathy by deep learning analysis of en face images of choroidal vasculature

PurposeTo classify central serous chorioretinopathy (CSC) by deep learning (DL) analyses of en face images of choroidal vasculature obtained by optical coherence tomography (OCT) and to analyze the regions of interest for DL from heatmaps. MethodsOne-hundred eyes were studied; 53 eyes with CSC and 47 normal eyes. Volume scans of 12x12 mm square were obtained at the same time as the OCT angiographic (OCTA) scans (Plex Elite 9000 Swept-Source OCT(R), Zeiss). High-quality en face images of the choroidal vasculature of the segmentation slab of one-half of the subfoveal choroidal thickness were created for the analyses. The entire 100 en face images were divided into 80 for training (100 times) and 20 for validation. The Neural Network Console (NNC) developed by Sony and the Keras-Tensorflow backend developed by Google were used as the software for the classification with 16 layers of convolutional neural networks. The active region of the heatmap based on the feature quantity extracted by DL was also evaluated as the percentages with gradient-weighted class activation mapping implementation in Keras. ResultsIn the 20 eyes used for validation including 8 eyes with CSC, the accuracy rate of the validation was 100% (20/20) for NNC and 95% (19/20) for Keras. This difference was not significant (P=0.33). The mean active region in the heatmap image was 12.5% in CSC eyes which was significantly lower than the 79.8% in normal eyes (P<0.01). ConclusionsCSC can be automatically classified with high accuracy from en face images of the choroidal vasculature by DLs with different programs, convolutional layer structures, and small data sets. Heatmap analyses showed that DL focused on the area occupied by the choroidal vessels and their uniformity. We conclude that DL can help in the diagnosis of CSC.

biophysics↗

bioRxiv2019

Corollary Discharge Promotes a Sustained Motor State in a Neural Circuit for Navigation

Animals exhibit behavioral and neural responses that persist on longer time scales than transient or fluctuating stimulus inputs. Here, we report that C. elegans uses feedback from the motor circuit to a sensory processing interneuron to sustain its motor state during thermotactic navigation. By imaging circuit activity in behaving animals, we show that a principal postsynaptic partner of the AFD thermosensory neuron, the AIY interneuron, encodes both temperature and motor state information. By optogenetic and genetic manipulation of this circuit, we demonstrate that the motor state representation in AIY is a corollary discharge signal. RIM, an interneuron that is connected with premotor interneurons, is required for this corollary discharge. Ablation of RIM eliminates the motor representation in AIY, allows thermosensory representations to reach downstream premotor interneurons, and reduces the animals ability to sustain forward movements during thermotaxis. We propose that feedback from the motor circuit to the sensory processing circuit underlies a positive feedback mechanism to generate persistent neural activity and sustained behavioral patterns in a sensorimotor transformation.

neuroscience↗

bioRxiv2017

A descending pathway through electrical coupling facilitates undulatory wave propagation in C. elegans

Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with B-type motor neurons that drive forward locomotion. We combined genetic analysis, optogenetic manipulation, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generated intrinsic rhythmic activity, constituting distributed central pattern generators. Second, AVB premotor interneurons drove bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrained the frequency of body oscillators, forcing coherent propagation of bending waves. Despite substantial anatomical differences between the worm motor circuit and those in higher model organisms, we uncovered converging principles that govern coordinated locomotion.\n\nSignificance StatementA deep understanding of the neural basis of motor behavior must integrate neuromuscular dynamics, mechanosensory feedback, as well as global command signals, to predict behavioral dynamics. Here, we report on an integrative approach to defining the circuit logic underlying coordinated locomotion in C. elegans. Our combined experimental and computational analysis revealed that (1) motor neurons in C. elegans could function as intrinsic oscillators; (2) Descending inputs and proprioceptive couplings work synergistically to facilitate the sequential activation of motor neuron activities, allowing bending waves to propagate efficiently along the body. Our work thus represents a key step towards an integrative view of animal locomotion.

neuroscience↗

bioRxiv2017

Excitatory Motor Neurons Function As Central Pattern Generators In An Anatomically Compressed Motor Circuit For Reverse Locomotion

Central pattern generators are cell- or network-driven oscillators that underlie motor rhythmicity. The existence and identity of C. elegans CPGs are unknown. Through cell ablation, electrophysiology, and calcium imaging, we identified oscillators for C. elegans reverse locomotion. We show that the cholinergic and excitatory class A motor neurons exhibit intrinsic and oscillatory activity, and such an activity can drive reverse locomotion without premotor interneurons. Regulation of their oscillatory activity, either through effecting the P/Q/N high voltage-activated calcium channel, an endogenous constituent of their intrinsic oscillation, or, via the dual regulation by descending premotor interneurons, determines the propensity, velocity, and sustention of reverse locomotion. Thus, the reversal motor neuron themselves serve as distributed local oscillators; regulation of their intrinsic activity controls the reversal motor state. These findings exemplify anatomic and functional compression: motor executors integrate the role of rhythm generation in a locomotor network that is constrained by small cell numbers.\n\nHighlightsO_LIThe class A motor neurons (A-MNs) intrinsically oscillate.\nC_LIO_LIHigh voltage-activated calcium channel is a constituent of A-MN oscillation.\nC_LIO_LIRegulation of A-MN oscillation by interneurons determines the reversal state.\nC_LIO_LIA-MNs integrate the role of multiple neuron types in large locomotor networks.\nC_LI

neuroscience↗