A nonsense TMEM43 variant leads to disruption of connexin-linked function and autosomal dominant auditory neuropathy spectrum disorder.

Genes that are primarily expressed in cochlear glia-like supporting cells (GLSs) have not been clearly associated with progressive deafness. Herein, we present a deafness locus mapped to chromosome 3p25.1 and an auditory neuropathy spectrum disorder (ANSD) gene, TMEM43, mainly expressed in GLSs. We identify p.(Arg372Ter) of TMEM43 by linkage analysis and exome sequencing in two large Asian families segregating ANSD, which is characterized by inability to discriminate speech despite preserved sensitivity to sound. The knock-in mouse with the p.(Arg372Ter) variant recapitulates a progressive hearing loss with histological abnormalities in GLSs. Mechanistically, TMEM43 interacts with the Connexin26 and Connexin30 gap junction channels, disrupting the passive conductance current in GLSs in a dominant-negative fashion when the p.(Arg372Ter) variant is introduced. Based on these mechanistic insights, cochlear implant was performed on three subjects, and speech discrimination was successfully restored. Our study highlights a pathological role of cochlear GLSs by identifying a deafness gene and its causal relationship with ANSD.

Amplifying Colossal Thermal Expansion of a Martensitic Molecular Crystal through Interlayer Shear-induced Side-chain Liberation.

Molecular crystals capable of colossal thermal expansion (TE) are fascinating owing to their substantial and continuous volume changes and reasonably linear responses to temperature. This makes them promising candidates for micromachine applications. Macroscopic motion is driven by subtle yet cooperative movements of molecules that respond to the thermal motions of dynamic functional units. The study of p-TIPS-DSB presented here offers a compelling case highlighting the relationship between the degree of dynamicity of functional units and TE behavior. In its α-phase, the p-TIPS-DSB crystal undergoes an irreversible martensitic transition to the ß-phase, accompanied by significant cooperative interlayer shear. This process substantially enhances the mobility of the side-chains driven by the increased free volume surrounding them. This nearly doubles the volumetric TE coefficient from 255.3 (10) to 444.9 (32) MK-1, particularly in the actuation direction from 175.0 (7) to 291.7 (20) MK-1, enabling about 4.5% elongation/contraction. As demonstrated here, p-TIPS-DSB exhibits a decent force density (> 1.4 × 107 N m-3) and precise motion control capabilities due to its hysteresis-free and non-abrupt TE nature. Furthermore, we demonstrated the limited operating distance of colossal TE materials can be amplified by utilizing levers, highlighting the high potential of these materials for use in micromachines.

Active role of glia-like supporting cells in the organ of Corti: Membrane proteins and their roles in hearing.

The organ of Corti, located in the cochlea in the inner ear, is one of the major sensory organs involved in hearing. The organ of Corti consists of hair cells, glia-like supporting cells, and the cochlear nerve, which work in harmony to receive sound from the outer ear and transmit auditory signals to the cochlear nucleus in the auditory ascending pathway. In this process, maintenance of the endocochlear potential, with a high potassium gradient and clearance of electrolytes and biochemicals in the inner ear, is critical for normal sound transduction. There is an emerging need for a thorough understanding of each cell type involved in this process to understand the sophisticated mechanisms of the organ of Corti. Hair cells have long been thought to be active, playing a primary role in the cochlea in actively detecting and transmitting signals. In contrast, supporting cells are thought to be silent and function to support hair cells. However, growing lines of evidence regarding the membrane proteins that mediate ionic movement in supporting cells have demonstrated that supporting cells are not silent, but actively play important roles in normal signal transduction. In this review, we summarize studies that characterize diverse membrane proteins according to the supporting cell subtypes involved in cochlear physiology and hearing. This review contributes to a better understanding of supporting cell functions and facilitates the development of potential therapeutic tools for hearing loss.

Excitation-Inhibition Imbalance Leads to Alteration of Neuronal Coherence and Neurovascular Coupling under Acute Stress.

A single stressful event can cause morphologic and functional changes in neurons and even malfunction of vascular systems, which can lead to acute stress disorder or post-traumatic stress disorder. However, there is a lack of evidence regarding how acute stress impacts neuronal activity, the concurrent vascular response, and the relationship between these two factors, which is defined as neurovascular coupling. Here, using in vivo two-photon imaging, we found that NMDA-evoked calcium transients of excitatory neurons were impaired and that vasodilation of penetrating arterioles was concomitantly disrupted in acutely stressed male mice. Furthermore, acute stress altered the relationship between excitatory neuronal calcium coherence and vascular responses. By measuring NMDA-evoked excitatory and inhibitory neuronal calcium activity in acute brain slices, we confirmed that neuronal coherence both between excitatory neurons and between excitatory and inhibitory neurons was reduced by acute stress but restored by blockade of glucocorticoid receptor signaling. Furthermore, the ratio of sEPSCs to sIPSCs was altered by acute stress, suggesting that the excitation-inhibition balance was disrupted by acute stress. In summary, in vivo, ex vivo, and whole-cell recording studies demonstrate that acute stress modifies excitatory-inhibitory neuronal coherence, disrupts the excitation-inhibition balance, and causes consequent neurovascular coupling changes, providing critical insights into the neural mechanism of stress-induced disorders.SIGNIFICANCE STATEMENT Acute stress can cause pathologic conditions, such as acute stress disorder and post-traumatic stress disorder, by affecting the functions of neurons and blood vessels. However, investigations into the impacts of acute stress on neurovascular coupling, the tight connection between local neural activity and subsequent blood flow changes, are lacking. Through investigations at the in vivo, ex vivo, and whole-cell recording levels, we found that acute stress alters the NMDA-evoked vascular response, impairs the function and coherence of excitatory and inhibitory neurons, and disrupts the excitatory and inhibitory balance. These novel findings provide insights into the relevance of the excitatory-inhibitory balance, neuronal coherence, and neurovascular coupling to stress-induced disorders.

The molecular mechanism of synaptic activity-induced astrocytic volume transient.

KEY POINTS: Neuronal activity causes astrocytic volume change via K+ uptake through TREK-1 containing two-pore domain potassium channels. The volume transient is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel. Intense neuronal activity is synaptically coupled with a physical change in astrocytes via volume transients. ABSTRACT: The brain volume changes dynamically and transiently upon intense neuronal activity through a tight regulation of ion concentrations and water movement across the plasma membrane of astrocytes. We have recently demonstrated that an intense neuronal activity and subsequent astrocytic AQP4-dependent volume transient are critical for synaptic plasticity and memory. We have also pharmacologically demonstrated a functional coupling between synaptic activity and the astrocytic volume transient. However, the precise molecular mechanisms of how intense neuronal activity and the astrocytic volume transient are coupled remain unclear. Here we utilized an intrinsic optical signal imaging technique combined with fluorescence imaging using ion sensitive dyes and molecular probes and electrophysiology to investigate the detailed molecular mechanisms in genetically modified mice. We report that a brief synaptic activity induced by a train stimulation (20 Hz, 1 s) causes a prolonged astrocytic volume transient (80 s) via K+ uptake through TREK-1 containing two-pore domain potassium (K2P) channels, but not Kir4.1 or NKCC1. This volume change is terminated by Cl- efflux through the Ca2+ -activated anion channel BEST1, but not the volume-regulated anion channel TTYH. The source of the Ca2+ required to open BEST1 appears to be the stretch-activated TRPA1 channel in astrocytes, but not IP3 R2. In summary, our study identifies several important astrocytic ion channels (AQP4, TREK-1, BEST1, TRPA1) as the key molecules leading to the neuronal activity-dependent volume transient in astrocytes. Our findings reveal new molecular and cellular mechanisms for the synaptic coupling of intense neuronal activity with a physical change in astrocytes via volume transients.

Benefit in physical function and quality of life to nonsurgical treatment of varicose veins: Pilot study.

BACKGROUND: Studies on varicose veins have focused its effects on physical function; however, whether nonsurgical treatments alter muscle oxygenation or physical function remains unclear. Moreover, the differences in such functions between individuals with varicose veins and healthy individuals remain unclear. AIM: To investigate changes in physical function and the quality of life (QOL) following nonsurgical treatment of patients with varicose veins and determine the changes in their muscle oxygenation during activity. METHODS: We enrolled 37 participants (those with varicose veins, n = 17; healthy individuals, n = 20). We performed the following measurements pre- and post-nonsurgical treatment in the varicose vein patients and healthy individuals: Calf muscle oxygenation during the two-minute step test, open eyes one-leg stance, 30 s sit-to-stand test, visual analog scale (VAS) for pain, Pittsburgh sleep quality index, physical activity assessment, and QOL assessment. RESULTS: Varicose veins patients and healthy individuals differ in most variables (physical function, sleep quality, and QOL). Varicose veins patients showed significant differences between pre- and post-nonsurgical treatment- results in the 30 sit-to-stand test [14.41 (2.45) to 16.35 (4.11), P = 0.018), two-minute step test [162.29 (25.98) to 170.65 (23.80), P = 0.037], VAS for pain [5.35 (1.90) to 3.88 (1.73), P = 0.004], and QOL [39.34 (19.98) to 26.69 (17.02), P = 0.005]; however, no significant difference was observed for muscle oxygenation. CONCLUSION: Nonsurgical treatment improved lower extremity function and QOL in varicose veins patients, bringing their condition close to that of healthy individuals. Future studies should include patients with severe varicose veins requiring surgery to confirm our findings.

Effect of information and communication technology-based smart care services for physical and cognitive functions in older adults living alone: A quasi-experimental study.

BACKGROUND: As the global population ages and the number of older adults living alone increases, societies face the responsibility of building new support systems and providing novel forms of care to ensure the independence and happiness of sick or frail older individuals. This quasi-experimental study examined the association between information and communication technology-based smart care services and the physical and cognitive functions of older individuals living alone. METHODS: This study used a suite of smart technologies (artificial intelligence speaker, radar sensor, and personalized exercise App.) and interventions tailored to the initial physical functional scores of the participants. A total of 176 participants were recruited and assigned, with 88 participants in the intervention group and 88 in the control group. The short physical performance battery (SPPB), the digit span test (DST), and the Korean mini-mental state examination (K-MMSE) were used to assess participants before and after 12 weeks. RESULTS: No significant differences in gender, age, or educational levels were observed between the intervention and control groups. After adjusting for baseline performance, analysis of covariance revealed that the intervention group exhibited better outcomes in the SPPB five-time chair stand score (adjusted score difference: 0.329; P = 0.044) and the backward DST (adjusted score difference: 0.472; P = 0.007), but had lower score of K-MMSE (adjusted score difference: -0.935; P = 0.021), indicating enhanced lower limb muscle strength and cognitive function in working memory. CONCLUSION: ICT-based smart care services, combined with personalized exercise interventions, significantly support the physical and cognitive health of solitary older individuals. This approach highlights the potential of integrating smart technology and targeted physical activity to foster the well-being of the aging population living alone.

Hippocampal astrocytes induce sex-dimorphic effects on memory.

Astrocytic receptors influence cognitive function and can promote behavioral deficits in disease. These effects may vary based on variables such as biological sex, but it is not known if the effects of astrocytic receptors are dependent on sex. We leveraged in vivo gene editing and chemogenetics to examine the roles of astrocytic receptors in spatial memory and other processes. We show that reductions in metabotropic glutamate receptor 3 (mGluR3), the main astrocytic glutamate receptor in adults, impair memory in females but enhance memory in males. Similarly, increases in astrocytic mGluR3 levels have sex-dependent effects and enhance memory in females. mGluR3 manipulations also alter spatial search strategies during recall in a sex-specific manner. In addition, acute chemogenetic stimulation of Gi/o-coupled or Gs-coupled receptors in hippocampal astrocytes induces bidirectional and sex-dimorphic effects on memory. Thus, astrocytes are sex-dependent modulators of cognitive function and may promote sex differences in aging and disease.

An advanced sodium-ion rechargeable battery based on a tin-carbon anode and a layered oxide framework cathode.

In this work we report the characteristics and performance of a sodium-ion battery based on a Sn-C anode and a Na(Ni(0.5)Mn(0.5))O(2) cathode. We show that both electrodes behave satisfactorily in terms of capacity delivery and cycle life when tested in sodium semicells. By coupling these electrodes in an electrolyte solution of sodium perchlorate in a mixture of propylene carbonate and 2 %vol of fluoroethylene carbonate, a sodium-ion battery showing promising electrochemical performance is obtained. This sodium ion battery in fact operates at an average voltage of 2.8 V, with a specific capacity of 120 mA h g(-1) and with a life extending to 50 cycles with minor capacity decays.

Nonlinear Associations between Physical Function, Physical Activity, Sleep, and Depressive Symptoms in Older Adults.

The purpose of this study is to examine how physical function, physical activity, and sleep are related to depressive symptoms in older adults using a nonlinear model. The participants were 283 Korean older adults aged 65 and older who met the study inclusion criteria. Depressive symptoms were measured using the shortened version of the Geriatric Depression Scale in Korean (SGDS-K). Physical activity and sleep time were objectively quantified by continuously monitoring participants over 20 consecutive days using a triaxial accelerometer. Physical function was evaluated using five distinct measurements: grip strength, gait speed, the Timed Up and Go Test (TUG), the Six-Minute Walk Test (SMWT), and the Five Times Sit to Stand Test (FTSST). The SMWT, gait speed, and MVPA exhibited a nonlinear relationship with depressive symptoms. However, other physical functions showed linear relationships. Also, sleep time showed a U-shaped trend starting at approximately 390 min. After adjusting for age, sex, drinking, and smoking in the logistic regression model, SMWT, MVPA, and sleep time were significantly associated with depressive symptoms. The outcomes highlight the importance of considering multiple factors in understanding depression among the elderly, particularly the intricate interactions between these elements and biological rhythms.

Health Provider's Feedback on Physical Activity Surveillance Using Wearable Device-Smartphone Application for Adults with Metabolic Syndrome; a 12-Week Randomized Control Study.

Purpose: Research on whether wearable device interventions can effectively prevent metabolic syndrome remains insufficient. This study aimed to evaluate the effect of feedback on clinical indicators in patients with metabolic syndrome on activities measured using wearable devices, such as smartphone apps. Methods: Patients with metabolic syndrome were recruited and prescribed to live for 12 weeks using a wrist-wearable device (B.BAND, B Life Inc., Korea). A block randomization method was used to distribute participants between the intervention (n=35) and control groups (n=32). In the intervention group, an experienced study coordinator provided feedback on physical activity to individuals through telephonic counseling every other week. Results: The mean number of steps in the control group was 8892.86 (4473.53), and those in the intervention group was 10,129.31 (4224.11). After 12 weeks, metabolic syndrome was resolved. Notably, there were statistically significant differences in the metabolic composition among the participants who completed the intervention. The mean number of metabolic disorder components per person remained at 3 in the control group, and decreased from 4 to 3 in the intervention group. Additionally, waist circumference, systolic and diastolic blood pressure, and triglyceride levels were significantly reduced, while HDL-cholesterol levels were significantly increased in the intervention group. Conclusion: Overall, 12 weeks of telephonic counseling intervention using wearable device-based physical activity confirmation improved the damaged metabolic components of patients with metabolic syndrome. Telephonic intervention can help increase physical activity and reduce waist circumference, which is a typical clinical indicator of metabolic syndrome.

Opto-vTrap, an optogenetic trap for reversible inhibition of vesicular release, synaptic transmission, and behavior.

Spatiotemporal control of brain activity by optogenetics has emerged as an essential tool to study brain function. For silencing brain activity, optogenetic probes, such as halorhodopsin and archaerhodopsin, inhibit transmitter release indirectly by hyperpolarizing membrane potentials. However, these probes cause an undesirable ionic imbalance and rebound spikes. Moreover, they are not applicable to use in non-excitable glial cells. Here we engineered Opto-vTrap, a light-inducible and reversible inhibition system to temporarily trap the transmitter-containing vesicles from exocytotic release. Light activation of Opto-vTrap caused full vesicle clusterization and complete inhibition of exocytosis within 1 min, which recovered within 30 min after light off. We found a significant reduction in synaptic and gliotransmission upon activation of Opto-vTrap in acute brain slices. Opto-vTrap significantly inhibited hippocampus-dependent memory retrieval with full recovery within an hour. We propose Opto-vTrap as a next-generation optogenetic silencer to control brain activity and behavior with minimal confounding effects.

Retina-attached slice recording reveals light-triggered tonic GABA signaling in suprachiasmatic nucleus.

Light is a powerful external cue modulating the biological rhythm of internal clock neurons in the suprachiasmatic nucleus (SCN). GABA signaling in SCN is critically involved in this process. Both phasic and tonic modes of GABA signaling exist in SCN. Of the two modes, the tonic mode of GABA signaling has been implicated in light-mediated synchrony of SCN neurons. However, modulatory effects of external light on tonic GABA signalling are yet to be explored. Here, we systematically characterized electrophysiological properties of the clock neurons and determined the spatio-temporal profiles of tonic GABA current. Based on the whole-cell patch-clamp recordings from 76 SCN neurons, the cells with large tonic GABA current (>15 pA) were more frequently found in dorsal SCN. Moreover, tonic GABA current in SCN was highly correlated with the frequency of spontaneous inhibitory postsynaptic current (sIPSC), raising a possibility that tonic GABA current is due to spill-over from synaptic release. Interestingly, tonic GABA current was inversely correlated with slice-to-patch time interval, suggesting a critical role of retinal light exposure in intact brain for an induction of tonic GABA current in SCN. To test this possibility, we obtained meticulously prepared retina-attached SCN slices and successfully recorded tonic and phasic GABA signaling in SCN neurons. For the first time, we observed an early-onset, long-lasting tonic GABA current, followed by a slow-onset, short-lasting increase in the phasic GABA frequency, upon direct light-illumination of the attached retina. This result provides the first evidence that external light cue can directly trigger both tonic and phasic GABA signaling in SCN cell. In conclusion, we propose tonic GABA as the key mediator of external light in SCN.

A Deafness Associated Protein TMEM43 Interacts with KCNK3 (TASK-1) Two-pore Domain K+ (K2P) Channel in the Cochlea.

The TMEM43 has been studied in human diseases such as arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC5) and auditory neuropathy spectrum disorder (ANSD). In the heart, the p.(Ser358Leu) mutation has been shown to alter intercalated disc protein function and disturb beating rhythms. In the cochlea, the p.(Arg372Ter) mutation has been shown to disrupt connexin-linked function in glia-like supporting cells (GLSs), which maintain inner ear homeostasis for hearing. The TMEM43-p.(Arg372Ter) mutant knock-in mice displayed a significantly reduced passive conductance current in the cochlear GLSs, raising a possibility that TMEM43 is essential for mediating the passive conductance current in GLSs. In the brain, the two-pore-domain potassium (K2P) channels are generally known as the "leak channels" to mediate background conductance current, raising another possibility that K2P channels might contribute to the passive conductance current in GLSs. However, the possible association between TMEM43 and K2P channels has not been investigated yet. In this study, we examined whether TMEM43 physically interacts with one of the K2P channels in the cochlea, KCNK3 (TASK-1). Utilizing co-immunoprecipitation (IP) assay and Duolink proximity ligation assay (PLA), we revealed that TMEM43 and TASK-1 proteins could directly interact. Genetic modifications further delineated that the intracellular loop domain of TMEM43 is responsible for TASK-1 binding. In the end, gene-silencing of Task-1 resulted in significantly reduced passive conductance current in GLSs. Together, our findings demonstrate that TMEM43 and TASK-1 form a protein-protein interaction in the cochlea and provide the possibility that TASK-1 is a potential contributor to the passive conductance current in GLSs.

Platycodin D, a natural component of Platycodon grandiflorum, prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion.

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.

Inhibitors of synaptic vesicle exocytosis reduce surface expression of postsynaptic glutamate receptors.

Bafilomycin A1, a vacuolar H+-ATPase inhibitor, and botulinum toxin B and tetanus toxin, both vesicle fusion inhibitors, are widely known exocytosis blockers that have been used to inhibit the presynaptic release of neurotransmitters. However, protein trafficking mechanisms, such as the insertion of postsynaptic receptors and astrocytic glutamate-releasing channels into the plasma membrane, also require exocytosis. In our previous study, exocytosis inhibitors reduced the surface expression of astrocytic glutamate-releasing channels. Here, we further investigated whether exocytosis inhibitors influence the surface expression of postsynaptic receptors. Using pH-sensitive superecliptic pHluorin (SEP)-tagged postsynaptic glutamate receptors, including GluA1, GluA2, GluN1, and GluN2A, we found that bafilomycin A1, botulinum toxin B, and/or tetanus toxin reduce the SEP fluorescence of SEP-GluA1, SEP-GluA2, SEP-GluN1, and SEP-GluN2A. These findings indicate that presynaptic vesicle exocytosis inhibitors also affect the postsynaptic trafficking machinery for surface expression. Finally, this study provides profound insights assembling presynaptic, postsynaptic and astrocytic viewpoints into the interpretation of the data obtained using these synaptic vesicle exocytosis inhibitors.

The Relationship between Chronotype, Physical Activity and the Estimated Risk of Dementia in Community-Dwelling Older Adults.

Our study examined the association between chronotype, daily physical activity, and the estimated risk of dementia in 170 community-dwelling older adults. Chronotype was assessed with the Horne-Östberg Morningness-Eveningness Questionnaire (MEQ). Daily physical activity (of over 3 METs) was measured with a tri-axial accelerometer. The Korean version of the Mini-Mental State Examination (K-MMSE) was used to measure the estimated risk of dementia. The evening chronotype, low daily physical activity, and dementia were positively associated with each other. The participants with low physical activity alongside evening preference had 3.05 to 3.67 times higher estimated risk of developing dementia, and participants with low physical activity and morning preference had 1.95 to 2.26 times higher estimated risk than those with high physical activity and morning preference. Our study design does not infer causation. Nevertheless, our findings suggest that chronotype and daily physical activity are predictors of the risk of having dementia in older adults aged 70 years and above.

The Effect of a Virtual Reality-Based Intervention Program on Cognition in Older Adults with Mild Cognitive Impairment: A Randomized Control Trial.

This study aimed to investigate the association between a virtual reality (VR) intervention program and cognitive, brain and physical functions in high-risk older adults. In a randomized controlled trial, we enrolled 68 individuals with mild cognitive impairment (MCI). The MCI diagnosis was based on medical evaluations through a clinical interview conducted by a dementia specialist. Cognitive assessments were performed by neuropsychologists according to standardized methods, including the Mini-Mental State Examination (MMSE) and frontal cognitive function: trail making test (TMT) A & B, and symbol digit substitute test (SDST). Resting state electroencephalogram (EEG) was measured in eyes open and eyes closed conditions for 5 minutes each, with a 19-channel wireless EEG device. The VR intervention program (3 times/week, 100 min each session) comprised four types of VR game-based content to improve the attention, memory and processing speed. Analysis of the subjects for group-time interactions revealed that the intervention group exhibited a significantly improved executive function and brain function at the resting state. Additionally, gait speed and mobility were also significantly improved between and after the follow-up. The VR-based training program improved cognitive and physical function in patients with MCI relative to controls. Encouraging patients to perform VR and game-based training may be beneficial to prevent cognitive decline.

Tweety-homolog (Ttyh) Family Encodes the Pore-forming Subunits of the Swelling-dependent Volume-regulated Anion Channel (VRACswell) in the Brain.

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl-- and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRACswell) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRACswell in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl- conductance (ICl,swell) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant ICl,swell with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as ICl,swell observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRACswell in the brain.