RESUMEN
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.
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Chronic low back pain (LBP) can severely affect daily physical activity. Aberrant osteoclast-mediated resorption leads to porous endplates for the sensory innervation to cause LBP. Here, we report that the expression of proton-activated chloride (PAC) channel is induced during osteoclast differentiation in the porous endplates via a RANKL-NFATc1 signaling pathway. Extracellular acidosis evokes robust PAC currents in osteoclasts. An acidic environment of porous endplates and elevated PAC activation-enhanced osteoclast fusion provoke LBP. Further, we find that genetic knockout of PAC gene Pacc1 significantly reduces endplate porosity and spinal pain in a mouse LBP model, but it does not affect bone development or homeostasis of bone mass in adult mice. Moreover, both osteoclast bone resorptive compartment environment and PAC traffic from the plasma membrane to endosomes to form an intracellular organelle Cl channel have low pH around 5.0. The low pH environment activates PAC channel to increase sialyltransferase St3gal1 expression and sialylation of TLR2 in initiation of osteoclast fusion. Aberrant osteoclast-mediated resorption is also found in most skeletal disorders, including osteoarthritis, ankylosing spondylitis, rheumatoid arthritis, heterotopic ossification, enthesopathy. Thus, elevated Pacc1 expression and PAC activity could be a potential therapeutic target for LBP and osteoclast-associated pain.
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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.
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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.
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Barrera Hematoencefálica , Células Endoteliales , Mutación con Ganancia de Función , Neuronas Motoras , Canales Catiónicos TRPV , Animales , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Neuronas Motoras/patología , Neuronas Motoras/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Ratones , Degeneración Nerviosa/patología , Degeneración Nerviosa/genética , Fenotipo , Médula Espinal/patología , Médula Espinal/metabolismoRESUMEN
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.
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Cognición , Histona Acetiltransferasas , Trombospondinas , Vía de Señalización Wnt , Animales , Ratones , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Histona Acetiltransferasas/deficiencia , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Ratones Noqueados , Plasticidad Neuronal , Trombospondinas/genética , Trombospondinas/metabolismoAsunto(s)
Astrocitos , Cocaína , Ácido gamma-Aminobutírico , Transmisión Sináptica , Cocaína/farmacología , RecompensaRESUMEN
Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.
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Neuralgia , Traumatismos de los Nervios Periféricos , Ratones , Animales , Microglía , Dicumarol/uso terapéutico , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Médula Espinal , Adenosina Trifosfato/farmacología , Proteínas de la MembranaRESUMEN
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.
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Canales de Cloruro , Protones , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Microscopía por Crioelectrón , Dominios Proteicos , Fosfatidilinositol 4,5-Difosfato/metabolismoRESUMEN
Following peripheral nerve injury, extracellular ATP-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.
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Addictive drugs increase ventral tegmental area (VTA) dopamine (DA) neuron activity through distinct cellular mechanisms, one of which involves disinhibition of DA neurons by inhibiting local GABA neurons. How drugs regulate VTA GABA neuron activity and drive addictive behaviors remains poorly understood. Here, we show that astrocytes control VTA GABA neuron activity in cocaine reward via tonic inhibition in mice. Repeated cocaine exposure potentiates astrocytic tonic GABA release through volume-regulated anion channels (VRACs) and augments tonic inhibition of VTA GABA neurons, thus downregulating their activities and disinhibiting nucleus accumbens (NAc) projecting DA neurons. Attenuation of tonic inhibition by either deleting Swell1 (Lrrc8a), the obligatory subunit of VRACs, in VTA astrocytes or disrupting δ subunit of GABAA receptors in VTA GABA neurons reduces cocaine-evoked changes in neuron activity, locomotion, and reward behaviors in mice. Together, our findings reveal the critical role of astrocytes in regulating the VTA local circuit and cocaine reward.
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Cocaína , Ratones , Animales , Cocaína/farmacología , Área Tegmental Ventral/fisiología , Astrocitos , Neuronas Dopaminérgicas , Receptores de GABA-A , Ácido gamma-Aminobutírico , Recompensa , Proteínas de la MembranaRESUMEN
Desensitization is a common property of membrane receptors, including ion channels. The newly identified proton-activated chloride (PAC) channel plays an important role in regulating the pH and size of organelles in the endocytic pathway, and is also involved in acid-induced cell death. However, how the PAC channel desensitizes is largely unknown. Here, we show by patch-clamp electrophysiological studies that PAC (also known as TMEM206/ASOR) undergoes pH-dependent desensitization upon prolonged acid exposure. Through structure-guided and comprehensive mutagenesis, we identified several residues critical for PAC desensitization, including histidine (H) 98, glutamic acid (E) 94, and aspartic acid (D) 91 at the extracellular extension of the transmembrane helix 1 (TM1), as well as E107, D109, and E250 at the extracellular domain (ECD)-transmembrane domain (TMD) interface. Structural analysis and molecular dynamic simulations revealed extensive interactions between residues at the TM1 extension and those at the ECD-TMD interface. These interactions likely facilitate PAC desensitization by stabilizing the desensitized conformation of TM1, which undergoes a characteristic rotational movement from the resting and activated states to the desensitized state. Our studies establish a new paradigm of channel desensitization in this ubiquitously expressed ion channel and pave the way for future investigation of its relevance in cellular physiology and disease.
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Cloruros , Protones , Cloruros/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Dominios ProteicosRESUMEN
Sensing the mechanical microenvironment is an essential aspect of all life, yet its mechanism remains poorly understood. In this issue of Neuron, Chi et al. reveal the role of astrocyte mechanosensitive Piezo1 channel in adult neurogenesis and cognitive function.
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Astrocitos , Canales Iónicos , Astrocitos/metabolismo , Encéfalo/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , NeurogénesisRESUMEN
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.
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Canales de Cloruro , Cloruros , Protones , Ácido Aspártico/química , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Fenómenos Electrofisiológicos , Histidina/química , Humanos , Concentración de Iones de Hidrógeno , MutagénesisRESUMEN
During vesicular acidification, chloride (Cl-), as the counterion, provides the electrical shunt for proton pumping by the vacuolar H+ ATPase. Intracellular CLC transporters mediate Cl- influx to the endolysosomes through their 2Cl-/H+ exchange activity. However, whole-endolysosomal patch-clamp recording also revealed a mysterious conductance releasing Cl- from the lumen. It remains unknown whether CLCs or other Cl- channels are responsible for this activity. Here, we show that the newly identified proton-activated Cl- (PAC) channel traffics from the plasma membrane to endosomes via the classical YxxL motif. PAC deletion abolishes the endosomal Cl- conductance, raises luminal Cl- level, lowers luminal pH, and increases transferrin receptor-mediated endocytosis. PAC overexpression generates a large endosomal Cl- current with properties similar to those of endogenous conductance, hypo-acidifies endosomal pH, and reduces transferrin uptake. We propose that the endosomal Cl- PAC channel functions as a low pH sensor and prevents hyper-acidification by releasing Cl- from the lumen.
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Agonistas de los Canales de Cloruro/metabolismo , Endocitosis/inmunología , Endosomas/metabolismo , Transferrina/metabolismo , HumanosRESUMEN
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.
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Canales de Cloruro/química , Canales de Cloruro/metabolismo , Microscopía por Crioelectrón , Activación del Canal Iónico , Técnicas de Placa-Clamp , Imagen Individual de Molécula , Aniones/metabolismo , Sitios de Unión , Canales de Cloruro/ultraestructura , Cloruros/metabolismo , Ácido Glutámico/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico , Lisina/metabolismo , Modelos Moleculares , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Protones , Rotación , Especificidad por SustratoRESUMEN
Animal models of Down syndrome (DS), trisomic for human chromosome 21 (HSA21) genes or orthologs, provide insights into better understanding and treatment options. The only existing transchromosomic (Tc) mouse DS model, Tc1, carries a HSA21 with over 50 protein coding genes (PCGs) disrupted. Tc1 is mosaic, compromising interpretation of results. Here, we "clone" the 34 MB long arm of HSA21 (HSA21q) as a mouse artificial chromosome (MAC). Through multiple steps of microcell-mediated chromosome transfer, we created a new Tc DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs that are expressed and regulatable. TcMAC21 recapitulates many DS phenotypes including anomalies in heart, craniofacial skeleton and brain, molecular/cellular pathologies, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete genetic mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research.
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Cromosomas Humanos Par 21/genética , Síndrome de Down/genética , Ratones Transgénicos/genética , Animales , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Cardiopatías Congénitas/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Trisomía/genética , Secuenciación Completa del GenomaRESUMEN
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.
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Fibroblastos/inmunología , Interferones/inmunología , Proteínas de la Membrana/inmunología , Nucleótidos Cíclicos/inmunología , Canales Aniónicos Dependientes del Voltaje/inmunología , Animales , Antivirales/inmunología , Antivirales/metabolismo , Efecto Espectador , Línea Celular , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Herpes Simple/inmunología , Herpes Simple/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Humanos , Interferones/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleótidos Cíclicos/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Nucleotidiltransferasas/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
Severe local acidosis causes tissue damage and pain, and is associated with many diseases, including cerebral and cardiac ischemia, cancer, infection, and inflammation. However, the molecular mechanisms of the cellular response to extracellular acidic environment are not fully understood. We recently identified a novel and evolutionarily conserved membrane protein, PAC (also known as PACC1 or TMEM206), encoding the proton-activated chloride (Cl-) channel, whose activity is widely observed in human cell lines. We demonstrated that genetic deletion of Pac abolished the proton-activated Cl- currents in mouse neurons and also attenuated the acid-induced neuronal cell death and brain damage after ischemic stroke. Here, we show that the proton-activated Cl- currents are also conserved in primary rat cortical neurons, with characteristics similar to those observed in human and mouse cells. Pac gene knockdown nearly abolished the proton-activated Cl- currents in rat neurons and reduced the neuronal cell death triggered by acid treatment. These data further support the notion that activation of the PAC channel and subsequent Cl- entry into neurons during acidosis play a pathogenic role in acidotoxicity and brain injury.