LRRC8 Proteins Form Volume-Regulated Anion Channels that Sense Ionic Strength.

The volume-regulated anion channel (VRAC) is activated when a cell swells, and it plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC and how the channel is gated by cell swelling are unknown. Here, we show that SWELL1 and up to four other LRRC8 subunits assemble into heterogeneous complexes of ∼800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers, as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (Γ) in the absence of an osmotic gradient activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore and that hypotonic stress can activate VRAC through a decrease in cytoplasmic Γ.

Transfer of cGAMP into Bystander Cells via LRRC8 Volume-Regulated Anion Channels Augments STING-Mediated Interferon Responses and Anti-viral Immunity.

The enzyme cyclic GMP-AMP synthase (cGAS) senses cytosolic DNA in infected and malignant cells and catalyzes the formation of 2'3'cGMP-AMP (cGAMP), which in turn triggers interferon (IFN) production via the STING pathway. Here, we examined the contribution of anion channels to cGAMP transfer and anti-viral defense. A candidate screen revealed that inhibition of volume-regulated anion channels (VRACs) increased propagation of the DNA virus HSV-1 but not the RNA virus VSV. Chemical blockade or genetic ablation of LRRC8A/SWELL1, a VRAC subunit, resulted in defective IFN responses to HSV-1. Biochemical and electrophysiological analyses revealed that LRRC8A/LRRC8E-containing VRACs transport cGAMP and cyclic dinucleotides across the plasma membrane. Enhancing VRAC activity by hypotonic cell swelling, cisplatin, GTPγS, or the cytokines TNF or interleukin-1 increased STING-dependent IFN response to extracellular but not intracellular cGAMP. Lrrc8e-/- mice exhibited impaired IFN responses and compromised immunity to HSV-1. Our findings suggest that cell-to-cell transmission of cGAMP via LRRC8/VRAC channels is central to effective anti-viral immunity.

SWELL1, a plasma membrane protein, is an essential component of volume-regulated anion channel.

Maintenance of a constant cell volume in response to extracellular or intracellular osmotic changes is critical for cellular homeostasis. Activation of a ubiquitous volume-regulated anion channel (VRAC) plays a key role in this process; however, its molecular identity in vertebrates remains unknown. Here, we used a cell-based fluorescence assay and performed a genome-wide RNAi screen to find components of VRAC. We identified SWELL1 (LRRC8A), a member of a four-transmembrane protein family with unknown function, as essential for hypotonicity-induced iodide influx. SWELL1 is localized to the plasma membrane, and its knockdown dramatically reduces endogenous VRAC currents and regulatory cell volume decrease in various cell types. Furthermore, point mutations in SWELL1 cause a significant change in VRAC anion selectivity, demonstrating that SWELL1 is an essential VRAC component. These findings enable further molecular characterization of the VRAC channel complex and genetic studies for understanding the function of VRAC in normal physiology and disease.

Structures and pH-sensing mechanism of the proton-activated chloride channel.

The proton-activated chloride channel (PAC) is active across a wide range of mammalian cells and is involved in acid-induced cell death and tissue injury1-3. PAC has recently been shown to represent a novel and evolutionarily conserved protein family4,5. Here we present two cryo-electron microscopy structures of human PAC in a high-pH resting closed state and a low-pH proton-bound non-conducting state. PAC is a trimer in which each subunit consists of a transmembrane domain (TMD), which is formed of two helices (TM1 and TM2), and an extracellular domain (ECD). Upon a decrease of pH from 8 to 4, we observed marked conformational changes in the ECD-TMD interface and the TMD. The rearrangement of the ECD-TMD interface is characterized by the movement of the histidine 98 residue, which is, after acidification, decoupled from the resting position and inserted into an acidic pocket that is about 5 Å away. Within the TMD, TM1 undergoes a rotational movement, switching its interaction partner from its cognate TM2 to the adjacent TM2. The anion selectivity of PAC is determined by the positively charged lysine 319 residue on TM2, and replacing lysine 319 with a glutamate residue converts PAC to a cation-selective channel. Our data provide a glimpse of the molecular assembly of PAC, and a basis for understanding the mechanism of proton-dependent activation.

Molecular determinants of pH sensing in the proton-activated chloride channel.

In response to acidic pH, the widely expressed proton-activated chloride (PAC) channel opens and conducts anions across cellular membranes. By doing so, PAC plays an important role in both cellular physiology (endosome acidification) and diseases associated with tissue acidosis (acid-induced cell death). Despite the available structural information, how proton binding in the extracellular domain (ECD) leads to PAC channel opening remains largely unknown. Here, through comprehensive mutagenesis and electrophysiological studies, we identified several critical titratable residues, including two histidine residues (H130 and H131) and an aspartic acid residue (D269) at the distal end of the ECD, together with the previously characterized H98 at the transmembrane domain-ECD interface, as potential pH sensors for human PAC. Mutations of these residues resulted in significant changes in pH sensitivity. Some combined mutants also exhibited large basal PAC channel activities at neutral pH. By combining molecular dynamics simulations with structural and functional analysis, we further found that the ß12 strand at the intersubunit interface and the associated "joint region" connecting the upper and lower ECDs allosterically regulate the proton-dependent PAC activation. Our studies suggest a distinct pH-sensing and gating mechanism of this new family of ion channels sensitive to acidic environment.

Dissociation of EphB2 signaling pathways mediating progenitor cell proliferation and tumor suppression.

Signaling proteins driving the proliferation of stem and progenitor cells are often encoded by proto-oncogenes. EphB receptors represent a rare exception; they promote cell proliferation in the intestinal epithelium and function as tumor suppressors by controlling cell migration and inhibiting invasive growth. We show that cell migration and proliferation are controlled independently by the receptor EphB2. EphB2 regulated cell positioning is kinase-independent and mediated via phosphatidylinositol 3-kinase, whereas EphB2 tyrosine kinase activity regulates cell proliferation through an Abl-cyclin D1 pathway. Cyclin D1 regulation becomes uncoupled from EphB signaling during the progression from adenoma to colon carcinoma in humans, allowing continued proliferation with invasive growth. The dissociation of EphB2 signaling pathways enables the selective inhibition of the mitogenic effect without affecting the tumor suppressor function and identifies a pharmacological strategy to suppress adenoma growth.

Piezo2 senses airway stretch and mediates lung inflation-induced apnoea.

Respiratory dysfunction is a notorious cause of perinatal mortality in infants and sleep apnoea in adults, but the mechanisms of respiratory control are not clearly understood. Mechanical signals transduced by airway-innervating sensory neurons control respiration; however, the physiological significance and molecular mechanisms of these signals remain obscured. Here we show that global and sensory neuron-specific ablation of the mechanically activated ion channel Piezo2 causes respiratory distress and death in newborn mice. Optogenetic activation of Piezo2+ vagal sensory neurons causes apnoea in adult mice. Moreover, induced ablation of Piezo2 in sensory neurons of adult mice causes decreased neuronal responses to lung inflation, an impaired Hering-Breuer mechanoreflex, and increased tidal volume under normal conditions. These phenotypes are reproduced in mice lacking Piezo2 in the nodose ganglion. Our data suggest that Piezo2 is an airway stretch sensor and that Piezo2-mediated mechanotransduction within various airway-innervating sensory neurons is critical for establishing efficient respiration at birth and maintaining normal breathing in adults.

What biologists want from their chloride reporters - a conversation between chemists and biologists.

Impaired chloride transport affects diverse processes ranging from neuron excitability to water secretion, which underlie epilepsy and cystic fibrosis, respectively. The ability to image chloride fluxes with fluorescent probes has been essential for the investigation of the roles of chloride channels and transporters in health and disease. Therefore, developing effective fluorescent chloride reporters is critical to characterizing chloride transporters and discovering new ones. However, each chloride channel or transporter has a unique functional context that demands a suite of chloride probes with appropriate sensing characteristics. This Review seeks to juxtapose the biology of chloride transport with the chemistries underlying chloride sensors by exploring the various biological roles of chloride and highlighting the insights delivered by studies using chloride reporters. We then delineate the evolution of small-molecule sensors and genetically encoded chloride reporters. Finally, we analyze discussions with chloride biologists to identify the advantages and limitations of sensors in each biological context, as well as to recognize the key design challenges that must be overcome for developing the next generation of chloride sensors.

Piezo2 is required for Merkel-cell mechanotransduction.

How we sense touch remains fundamentally unknown. The Merkel cell-neurite complex is a gentle touch receptor in the skin that mediates slowly adapting responses of Aß sensory fibres to encode fine details of objects. This mechanoreceptor complex was recognized to have an essential role in sensing gentle touch nearly 50 years ago. However, whether Merkel cells or afferent fibres themselves sense mechanical force is still debated, and the molecular mechanism of mechanotransduction is unknown. Synapse-like junctions are observed between Merkel cells and associated afferents, and yet it is unclear whether Merkel cells are inherently mechanosensitive or whether they can rapidly transmit such information to the neighbouring nerve. Here we show that Merkel cells produce touch-sensitive currents in vitro. Piezo2, a mechanically activated cation channel, is expressed in Merkel cells. We engineered mice deficient in Piezo2 in the skin, but not in sensory neurons, and show that Merkel-cell mechanosensitivity completely depends on Piezo2. In these mice, slowly adapting responses in vivo mediated by the Merkel cell-neurite complex show reduced static firing rates, and moreover, the mice display moderately decreased behavioural responses to gentle touch. Our results indicate that Piezo2 is the Merkel-cell mechanotransduction channel and provide the first line of evidence that Piezo channels have a physiological role in mechanosensation in mammals. Furthermore, our data present evidence for a two-receptor-site model, in which both Merkel cells and innervating afferents act together as mechanosensors. The two-receptor system could provide this mechanoreceptor complex with a tuning mechanism to achieve highly sophisticated responses to a given mechanical stimulus.

Piezo2 is the major transducer of mechanical forces for touch sensation in mice.

The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell-neurite complexes. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron; however, major aspects of touch sensation remain intact without Merkel cell activity. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.

Molecular Biology and Physiology of Volume-Regulated Anion Channel (VRAC).

The Volume-Regulated Anion Channel (VRAC) is activated by cell swelling and plays a key role in cell volume regulation. VRAC is ubiquitously expressed in vertebrate cells and also implicated in many other physiological and cellular processes including fluid secretion, glutamate release, membrane potential regulation, cell proliferation, migration, and apoptosis. Although its biophysical properties have been well characterized, the molecular identity of VRAC remained a mystery for almost three decades. The field was transformed by recent discoveries showing that the leucine-rich repeat-containing protein 8A (LRRC8A, also named SWELL1) and its four other homologs form heteromeric VRAC channels. The composition of LRRC8 subunits determines channel properties and substrate selectivity of a large variety of different VRACs. Incorporating purified SWELL1-containing protein complexes into lipid bilayers is sufficient to reconstitute channel activities, a finding that supports the decrease in intracellular ionic strength as the mechanism of VRAC activation during cell swelling. Characterization of Swell1 knockout mice uncovers the important role of VRAC in T cell development, pancreatic ß-cell glucose-stimulated insulin secretion, and adipocyte metabolic function. The ability to permeate organic osmolytes and metabolites is a major feature of VRAC. The list of VRAC substrates is expected to grow, now also including some cancer drugs and antibiotics even under non-cell swelling conditions. Therefore, a critical role of VRAC in drug resistance and cell-cell communication is emerging. This review summarizes the exciting recent progress on the structure-function relationship and physiology of VRAC and discusses key future questions to be solved.

Piezo1, a mechanically activated ion channel, is required for vascular development in mice.

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.

KAT6A deficiency impairs cognitive functions through suppressing RSPO2/Wnt signaling in hippocampal CA3.

Intellectual disability (ID) affects ~2% of the population and ID-associated genes are enriched for epigenetic factors, including those encoding the largest family of histone lysine acetyltransferases (KAT5-KAT8). Among them is KAT6A, whose mutations cause KAT6A syndrome, with ID as a common clinical feature. However, the underlying molecular mechanism remains unknown. Here, we find that KAT6A deficiency impairs synaptic structure and plasticity in hippocampal CA3, but not in CA1 region, resulting in memory deficits in mice. We further identify a CA3-enriched gene Rspo2, encoding Wnt activator R-spondin 2, as a key transcriptional target of KAT6A. Deletion of Rspo2 in excitatory neurons impairs memory formation, and restoring RSPO2 expression in CA3 neurons rescues the deficits in Wnt signaling and learning-associated behaviors in Kat6a mutant mice. Collectively, our results demonstrate that KAT6A-RSPO2-Wnt signaling plays a critical role in regulating hippocampal CA3 synaptic plasticity and cognitive function, providing potential therapeutic targets for KAT6A syndrome and related neurodevelopmental diseases.

The SWELL1 Channel Promotes Ischemic Brain Damage by Mediating Neuronal Swelling and Glutamate Toxicity.

Cytotoxic neuronal swelling and glutamate excitotoxicity are two hallmarks of ischemic stroke. However, the underlying molecular mechanisms are not well understood. Here, it is reported that SWELL1, the essential subunit of the volume-regulated anion channel (VRAC), plays a dual role in ischemic injury by promoting neuronal swelling and glutamate excitotoxicity. SWELL1 expression is upregulated in neurons and astrocytes after experimental stroke in mice. The neuronal SWELL1 channel is activated by intracellular hypertonicity, leading to Cl- influx-dependent cytotoxic neuronal swelling and subsequent cell death. Additionally, the SWELL1 channel in astrocytes mediates pathological glutamate release, indicated by increases in neuronal slow inward current frequency and tonic NMDAR current. Pharmacologically, targeting VRAC with a new inhibitor, an FDA-approved drug Dicumarol, attenuated cytotoxic neuronal swelling and cell death, reduced astrocytic glutamate release, and provided significant neuroprotection in mice when administered either before or after ischemia. Therefore, these findings uncover the pleiotropic effects of the SWELL1 channel in neurons and astrocytes in the pathogenesis of ischemic stroke and provide proof of concept for therapeutically targeting it in this disease.

Proton-activated chloride channel increases endplate porosity and pain in a mouse spine degeneration model.

Chronic low back pain (LBP) can severely affect daily physical activity. Aberrant osteoclast-mediated resorption leads to porous endplates, which allow the sensory innervation that causes LBP. Here, we report that expression of the proton-activated chloride (PAC) channel was induced during osteoclast differentiation in the porous endplates via a RANKL/NFATc1 signaling pathway. Extracellular acidosis evoked robust PAC currents in osteoclasts. An acidic environment of porous endplates and elevated PAC activation-enhanced osteoclast fusion provoked LBP. Furthermore, we found that genetic knockout of the PAC gene Pacc1 significantly reduced endplate porosity and spinal pain in a mouse LBP model, but it did not affect bone development or homeostasis of bone mass in adult mice. Moreover, both the osteoclast bone-resorptive compartment environment and PAC traffic from the plasma membrane to endosomes to form an intracellular organelle Cl channel had a low pH of approximately 5.0. The low pH environment activated the PAC channel to increase sialyltransferase St3gal1 expression and sialylation of TLR2 in the initiation of osteoclast fusion. Aberrant osteoclast-mediated resorption is also found in most skeletal disorders, including osteoarthritis, ankylosing spondylitis, rheumatoid arthritis, heterotopic ossification, and enthesopathy. Thus, elevated Pacc1 expression and PAC activity could be a potential therapeutic target for the treatment of LBP and osteoclast-associated pain.

Tunable Cytosolic Chloride Indicators for Real-Time Chloride Imaging in Live Cells.

Chloride plays a crucial role in various cellular functions, and its level is regulated by a variety of chloride transporters and channels. However, to date, we still lack the capability to image instantaneous ion flux through chloride channels at single-cell level. Here, we developed a series of cell-permeable, pH-independent, chloride-sensitive fluorophores for real-time cytosolic chloride imaging, which we call CytoCl dyes. We demonstrated the ability of CytoCl dyes to monitor cytosolic chloride and used it to uncover the rapid changes and transient events of halide flux, which cannot be captured by steady-state imaging. Finally, we successfully imaged the proton-activated chloride channel-mediated ion flux at single-cell level, which is, to our knowledge, the first real-time imaging of ion flux through a chloride channel in unmodified cells. By enabling the imaging of single-cell level ion influx through chloride channels and transporters, CytoCl dyes can expand our understanding of ion flux dynamics, which is critical for characterization and modulator screening of these membrane proteins. A conjugable version of CytoCl dyes was also developed for its customization across different applications.

Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice.

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.

c-Abl tyrosine kinase regulates cardiac growth and development.

The c-Abl protein is a ubiquitously expressed nonreceptor tyrosine kinase involved in the development and function of many mammalian organ systems, including the immune system and bone. Here we show that homozygous Abl mutant embryos and newborns on the C57BL/6J background, but not on other backgrounds, display dramatically enlarged hearts and die perinatally. The heart defects can be largely rescued by cardiomyocyte-specific restoration of the full-length c-Abl protein. The cardiac hyperplasia phenotype is not caused by decreased apoptosis, but rather by abnormally increased cardiomyocyte proliferation during later stages of embryogenesis. Genes involved in cardiac stress and remodeling and cell cycle regulation are also up-regulated in the mutant hearts. These findings reveal an essential role for c-Abl in mammalian heart growth and development.

Inhibition of the proton-activated chloride channel PAC by PIP2.

Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by PACC1 (TMEM206). PAC regulates endosomal acidification and macropinosome shrinkage by releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP2) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP2 at pH 4.0 and identified its putative binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP2-binding. Structural and electrophysiological analyses suggest that PIP2 inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP2 as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.