ABSTRACT
Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits.
Subject(s)
Epilepsy , Voltage-Gated Sodium Channels , Humans , Neurons/metabolism , Voltage-Gated Sodium Channels/metabolism , Epilepsy/metabolism , Brain/metabolism , Seizures/drug therapy , Seizures/metabolism , Action Potentials/physiologyABSTRACT
Moyamoya disease, a cerebrovascular disease leading to strokes in children and young adults, is characterized by progressive occlusion of the distal internal carotid arteries and the formation of collateral vessels. Altered genes play a prominent role in the aetiology of moyamoya disease, but a causative gene is not identified in the majority of cases. Exome sequencing data from 151 individuals from 84 unsolved families were analysed to identify further genes for moyamoya disease, then candidate genes assessed in additional cases (150 probands). Two families had the same rare variant in ANO1, which encodes a calcium-activated chloride channel, anoctamin-1. Haplotype analyses found the families were related, and ANO1 p.Met658Val segregated with moyamoya disease in the family with an LOD score of 3.3. Six additional ANO1 rare variants were identified in moyamoya disease families. The ANO1 rare variants were assessed using patch-clamp recordings, and the majority of variants, including ANO1 p.Met658Val, displayed increased sensitivity to intracellular Ca2+. Patients harbouring these gain-of-function ANO1 variants had classic features of moyamoya disease, but also had aneurysm, stenosis and/or occlusion in the posterior circulation. Our studies support that ANO1 gain-of-function pathogenic variants predispose to moyamoya disease and are associated with unique involvement of the posterior circulation.
Subject(s)
Anoctamin-1 , Moyamoya Disease , Child , Humans , Young Adult , Anoctamin-1/genetics , Chloride Channels/genetics , Moyamoya Disease/genetics , Neoplasm Proteins/geneticsABSTRACT
Secondary ovarian tumor [secondary tumor of the ovary (STO)] is not a frequent disease. To date, there is still a lack of standard treatment for STO due to the relative heterogeneity. Liver cancer metastasis to the ovary is extremely rare, with only 17 living cases having been reported so far, making it impossible to launch large-scale prospective studies and formulate the standard intervention for patients. We herein report a rare case of STO with liver primary cancer metastasis to the ovary and omentum in a 66-year-old woman. The patient underwent debulking surgery with the removal of the uterus, bilateral fallopian tubes, bilateral ovaries, appendix, and a large part of the omentum majus. Next-generation sequencing was conducted after the operation, identifying BRCA2 mutation. Because strongly refusing chemotherapy, she received olaparib as an experimental therapy. After the administration of surgery and olaparib, the serum value of cancer antigen 125 (CA125) and alpha fetoprotein (AFP) decreased dramatically and basically remained within the normal range. So far, she has achieved nearly 2-year survival and lives a relatively normal life with good quality.
ABSTRACT
The voltage-gated ion channel activity depends on both activation (transition from the resting state to the open state) and inactivation. Inactivation is a self-restraint mechanism to limit ion conduction and is as crucial to membrane excitability as activation. Inactivation can occur when the channel is open or closed. Although open-state inactivation is well understood, the molecular basis of closed-state inactivation has remained elusive. We report cryo-EM structures of human KV4.2 channel complexes in inactivated, open, and closed states. Closed-state inactivation of KV4 involves an unprecedented symmetry breakdown for pore closure by only two of the four S4-S5 linkers, distinct from known mechanisms of open-state inactivation. We further capture KV4 in a putative resting state, revealing how voltage sensor movements control the pore. Moreover, our structures provide insights regarding channel modulation by KChIP2 and DPP6 auxiliary subunits. Our findings elucidate mechanisms of closed-state inactivation and voltage-dependent activation of the KV4 channel.
Subject(s)
Ion Channel Gating , Shal Potassium Channels , Humans , Ion Channel Gating/physiology , Kinetics , Membrane Potentials/physiology , Shal Potassium Channels/genetics , Shal Potassium Channels/metabolismABSTRACT
Ovarian aging is a pacemaker with multiple organ dysfunction. Recently, stem cells with the ability to generate new oocytes have been identified, which provides the possibility of stem cell therapy for ovarian aging. Several studies have revealed the existence of stem cells in the human postmenopausal ovary. In this study, we describe a new method using magnetic-activated cell sorting combined with differential adhesion to isolate DDX4+ stem cells from ovaries of postmenopausal women and show that the cells exhibit similar gene expression profiles and growth characteristics with primitive germ cells. Furthermore, the DDX4+ stem cells could enter the meiosis stage and differentiation into oocytes. The RNA-seq data of the differentiated oocytes shows that mitochondrial metabolism may play an important role in the oogenesis process of the DDX4+ stem cells. Through using the human ovarian cortical fragments transplantation model, we indicated that the GFP-DDX4+ stem cells differentiated into some GFP positive oocyte-like structure in vivo. Our study provided a new method for the isolation of DDX4+ stem cells from the ovaries of postmenopausal women and confirmed the ability of these stem cells to differentiate into oocytes.
Subject(s)
Ovary , Postmenopause , Cell Differentiation , Female , Germ Cells , Humans , Oocytes , Ovary/metabolism , Stem Cells/metabolismABSTRACT
The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently1. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions2-5. MRGPRX2 couples to both Gi and Gq in mast cells6. Here we describe agonist-stabilized structures of MRGPRX2 coupled to Gi1 and Gq in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and Gq. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.
Subject(s)
Cryoelectron Microscopy , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/chemistry , Pruritus/metabolism , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Receptors, Neuropeptide/antagonists & inhibitors , Receptors, Neuropeptide/chemistry , Drug Inverse Agonism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/ultrastructure , GTP-Binding Protein alpha Subunits, Gq-G11/chemistry , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/ultrastructure , Humans , Models, Molecular , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/ultrastructure , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/ultrastructure , Receptors, Neuropeptide/metabolism , Receptors, Neuropeptide/ultrastructureABSTRACT
The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.
Subject(s)
Anoctamin-1/metabolism , Neoplasm Proteins/metabolism , Respiratory Mucosa/embryology , Animals , Cell Differentiation/physiology , Humans , Mice , Organogenesis/physiology , Respiratory Mucosa/metabolism , Trachea/embryology , Trachea/metabolismABSTRACT
TMEM16B (ANO2) is the Ca2+-activated chloride channel expressed in multiple brain regions, including the amygdala. Here we report that Ano2 knockout mice exhibit impaired anxiety-related behaviors and context-independent fear memory, thus implicating TMEM16B in anxiety modulation. We found that TMEM16B is expressed in somatostatin-positive (SOM+) GABAergic neurons of the central lateral amygdala (CeL), and its activity modulates action potential duration and inhibitory postsynaptic current (IPSC). We further provide evidence for TMEM16B actions not only in the soma but also in the presynaptic nerve terminals of GABAergic neurons. Our study reveals an intriguing role for TMEM16B in context-independent but not context-dependent fear memory, and supports the notion that dysfunction of the amygdala contributes to anxiety-related behaviors.
Subject(s)
Amygdala/physiology , Anoctamins/metabolism , Anxiety , GABAergic Neurons/physiology , Signal Transduction , Animals , Anoctamins/deficiency , Behavior, Animal , Mice, KnockoutABSTRACT
TMEM16F is activated by elevated intracellular Ca2+, and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.
Subject(s)
Anions/metabolism , Anoctamins/chemistry , Anoctamins/metabolism , Cations/metabolism , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/metabolism , Animals , Anoctamins/genetics , DNA Mutational Analysis , Mice , Phospholipid Transfer Proteins/genetics , Substrate SpecificityABSTRACT
As a Ca2+-activated lipid scramblase and ion channel that mediates Ca2+ influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP2-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca2+ influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation.
ABSTRACT
There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function.
Subject(s)
Aging/genetics , Ovarian Follicle/growth & development , Ovary/growth & development , RNA/genetics , Animals , DNA Repair/genetics , DNA Replication/genetics , Female , Gene Expression Regulation, Developmental/genetics , Humans , Menopause/genetics , Mice , Oocytes/growth & development , Oocytes/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Ovulation/genetics , RNA/biosynthesis , Transcriptome/geneticsABSTRACT
Chemotherapy-induced ovarian aging not only increases the risk for early menopause-related complications but also results in infertility in young female cancer survivors. Oogonial stem cells have the ability to generate new oocytes and thus provide new opportunities for treating ovarian aging and female infertility. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phenol derived from plants, that has been shown to have positive effects on longevity and redox flow in lipid metabolism and a preventive function against certain tumors. To evaluate whether resveratrol could promote the repair of oogonial stem cells damage in a busulfan/cyclophosphamide (Bu/Cy)-induced accelerated ovarian aging model, female mice were administered 30 and 100 mg/kg/d resveratrol through a gavage for 2 weeks. We demonstrated that resveratrol (30 mg/kg/d) relieved oogonial stem cells loss and showed an attenuating effect on Bu/Cy-induced oxidative apoptosis in mouse ovaries, which may be attributed to the attenuation of oxidative levels in ovaries. Additionally, we also showed that Res exerted a dose-dependent effect on oogonial stem cells and attenuated H2O2-induced cytotoxicity and oxidative stress injury by activating Nrf2 in vitro. Therefore, resveratrol could be of a potential therapeutic drug used to prevent chemotherapy-induced ovarian aging.
ABSTRACT
Calcium-activated phospholipid scramblase mediates the energy-independent bidirectional translocation of lipids across the bilayer, leading to transient or, in the case of apoptotic scrambling, sustained collapse of membrane asymmetry. Cells lacking TMEM16F-dependent lipid scrambling activity are deficient in generation of extracellular vesicles (EVs) that shed from the plasma membrane in a Ca2+-dependent manner, namely microvesicles. We have adapted chemical induction of giant plasma membrane vesicles (GPMVs), which require both TMEM16F-dependent phospholipid scrambling and calcium influx, as a kinetic assay to investigate the mechanism of TMEM16F activity. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that elucidate the mechanism for TMEM16F activation and facilitate further investigation of TMEM16F-mediated lipid translocation and its role in extracellular vesiculation.
Subject(s)
Anoctamins/metabolism , Biological Transport/physiology , Phospholipid Transfer Proteins/metabolism , Animals , Calcium/metabolism , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Cell-Derived Microparticles/metabolism , Extracellular Vesicles/metabolism , HEK293 Cells , Humans , Mice , Phospholipids/metabolismABSTRACT
The nociceptor ion channel TRPA1 detects a wide range of hazardous chemicals, including reactive electrophiles such as cinnamaldehyde, which gate the channel allowing Na+ and Ca2+ entry. TRPA1 assembles as a tetramer, with a central pore within which an aspartate residue (D918) determines Ca2+ permeability. Here, we report that introduction of histidine at this position, D918H, makes TRPA1 channels sensitive to block by nanomolar concentration of Zn2+ and can be used to functionally tag subunits in concatemers. Concatemers with increasing numbers of D918H subunits display increasing sensitivity to Zn2+ inhibition, indicating that the four side chains at position 918 of the tetramer directly coordinate Zn2+ and other permeating divalent cations. In the published structure of TRPA1, this requires a rearrangement of the pore region which may represent the true open state of the channel. Concatemeric channels containing subunits mutated to be insensitive to reactive electrophiles (C622S) could be activated by cinnamaldehyde when as few as two subunits contained intact ligand binding sites. Activation upon liganding of just two of the four possible subunits may represent an optimal strategy to rapidly and reliably detect noxious chemicals.
Subject(s)
Aspartic Acid/metabolism , Calcium/metabolism , Ion Channel Gating , Mutation , TRPA1 Cation Channel/metabolism , Zinc/metabolism , Acids/chemistry , Animals , Humans , Permeability , Protein Conformation , Protein Multimerization , Rats , TRPA1 Cation Channel/chemistry , TRPA1 Cation Channel/geneticsABSTRACT
TMEM16F, which is activated by elevation of intracellular calcium to trigger phospholipid scrambling and the collapse of lipid bilayer asymmetry to mediate important cellular functions such as blood coagulation, also generates a small-conductance calcium-activated cation current. How TMEM16F activation may be regulated is an open question. By recording TMEM16F Ca2+-activated current, we found that the TMEM16F Ca2+-response is desensitized by a brief exposure to high intracellular Ca2+, which is associated with depletion of phosphatidylinositol-(4, 5)-bisphosphate (PIP2) from the inner leaflet of the membrane. Application of artificial or natural PIP2 restores TMEM16F channel activity. PIP2 modulation of TMEM16F requires the presence of several positively charged amino acids in its cytoplasmic N-terminal domain. TMEM16F interaction with PIP2 works synergistically with membrane depolarization to facilitate Ca2+-gating of TMEM16F. Our study reveals the dependence of TMEM16F activity on phosphoinositides and provides one mechanism for TMEM16F activation to be strictly regulated in the cell membrane.
Subject(s)
Anoctamins/metabolism , Calcium/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phospholipid Transfer Proteins/metabolism , Animals , Anoctamins/chemistry , Anoctamins/genetics , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Humans , Mice , Phospholipid Transfer Proteins/chemistry , Phospholipid Transfer Proteins/genetics , Protein DomainsABSTRACT
Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, Otop2 and Otop3, and a Drosophila ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology.
Subject(s)
Ion Channels/genetics , Ion Channels/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Taste Buds/metabolism , Animals , Conserved Sequence , Drosophila melanogaster , Evolution, Molecular , HEK293 Cells , Humans , Ion Channels/classification , Membrane Proteins/classification , Mice , Otolithic Membrane/growth & development , Phylogeny , Protons , Tissue Distribution , TranscriptomeABSTRACT
Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.
Subject(s)
Anoctamin-1/chemistry , Anoctamin-1/ultrastructure , Calcium/chemistry , Calcium/pharmacology , Cryoelectron Microscopy , Ion Channel Gating/drug effects , Animals , Anions/chemistry , Anions/metabolism , Anoctamin-1/metabolism , Calcium/metabolism , Glucosides/chemistry , HEK293 Cells , Humans , Ion Transport/drug effects , Mice , Models, Molecular , Nanostructures/chemistry , Nanostructures/ultrastructure , Protein Conformation/drug effectsABSTRACT
Chloride is the major free anion in the extracellular space (>100 mM) and within the cytoplasm in eukaryotes (10 â¼ 20 mM). Cytoplasmic Cl- level is dynamically regulated by Cl- channels and transporters. It is well established that movement of Cl- across the cell membrane is coupled with cell excitability through changes in membrane potential and with water secretion. However, whether cytoplasmic Cl- plays additional roles in animal development and tissue homeostasis is unknown. Here we use genetics, cell biological and pharmacological tools to demonstrate that TMEM16A, an evolutionarily conserved calcium-activated chloride channel (CaCC), regulates cytoplasmic Cl- homeostasis and promotes plasma membrane remodeling required for mammalian epithelial morphogenesis. We demonstrate that TMEM16A-mediated control of cytoplasmic Cl- regulates the organization of the major phosphoinositide species PtdIns(4,5)P2 into microdomains on the plasma membrane, analogous to processes that cluster soluble and membrane proteins into phase-separated droplets. We further show that an adequate cytoplasmic Cl- level is required for proper endocytic trafficking and membrane supply during early stages of ciliogenesis and adherens junction remodeling. Our study thus uncovers a critical function of CaCC-mediated cytoplasmic Cl- homeostasis in controlling the organization of PtdIns(4,5)P2 microdomains and membrane remodeling. This newly defined role of cytoplasmic Cl- may shed light on the mechanisms of intracellular Cl- signaling events crucial for regulating tissue architecture and organelle biogenesis during animal development.