RESUMEN
Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.
Asunto(s)
Arabidopsis , Señalización del Calcio , Calcio , Germinación , Concentración Osmolar , Polen , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Germinación/genética , Mutación , Polen/genética , Polen/metabolismo , Agua/metabolismo , Células HEK293 , Humanos , DeshidrataciónRESUMEN
Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:
Asunto(s)
Coagulación Sanguínea , Plaquetas/metabolismo , Calcio/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Ambystoma mexicanum , Animales , Anoctamina-1 , Anoctaminas , Canales de Cloruro/metabolismo , Hemostasis , Metabolismo de los Lípidos , Megacariocitos/metabolismo , Ratones , Ratones Noqueados , Oocitos/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/genética , XenopusRESUMEN
Advancing the mechanistic understanding of absence epilepsy is crucial for developing new therapeutics, especially for patients unresponsive to current treatments. Utilizing a recently developed mouse model of absence epilepsy carrying the BK gain-of-function channelopathy D434G, here we report that attenuating the burst firing of midline thalamus (MLT) neurons effectively prevents absence seizures. We found that enhanced BK channel activity in the BK-D434G MLT neurons promotes synchronized bursting during the ictal phase of absence seizures. Modulating MLT neurons through pharmacological reagents, optogenetic stimulation, or deep brain stimulation effectively attenuates burst firing, leading to reduced absence seizure frequency and increased vigilance. Additionally, enhancing vigilance by amphetamine, a stimulant medication, or physical perturbation also effectively suppresses MLT bursting and prevents absence seizures. These findings suggest that the MLT is a promising target for clinical interventions. Our diverse approaches offer valuable insights for developing next generation therapeutics to treat absence epilepsy.
Asunto(s)
Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia , Animales , Epilepsia Tipo Ausencia/fisiopatología , Ratones , Tálamo/fisiopatología , Neuronas/metabolismo , Neuronas/fisiología , Optogenética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Estimulación Encefálica Profunda/métodos , Masculino , Núcleos Talámicos de la Línea Media/fisiologíaRESUMEN
TMEM16F (also known as ANO6), a Ca2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell-cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.
Asunto(s)
Anoctaminas , Células Endoteliales , Transducción de Señal , Animales , Anoctaminas/metabolismo , Anoctaminas/genética , Ratones , Humanos , Células Endoteliales/metabolismo , Familia-src Quinasas/metabolismo , Familia-src Quinasas/genética , Neovascularización Fisiológica , Fosforilación , Cadherinas/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Membrana Celular/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Angiogénesis , Proteínas de Transferencia de FosfolípidosRESUMEN
ABSTRACT: Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.
Asunto(s)
Anemia Hemolítica Congénita , Calcio , Femenino , Humanos , Anemia Hemolítica Congénita/genética , Calcio/metabolismo , Eritrocitos/metabolismo , Hidropesía Fetal/genética , Canales Iónicos/genética , Proteínas de Transferencia de Fosfolípidos/genéticaRESUMEN
A growing number of gain-of-function (GOF) BK channelopathies have been identified in patients with epilepsy and movement disorders. Nevertheless, the underlying pathophysiology and corresponding therapeutics remain obscure. Here, we utilized a knock-in mouse model carrying human BK-D434G channelopathy to investigate the neuronal mechanism of BK GOF in the pathogenesis of epilepsy and dyskinesia. The BK-D434G mice manifest the clinical features of absence epilepsy and exhibit severe motor deficits and dyskinesia-like behaviors. The cortical pyramidal neurons and cerebellar Purkinje cells from the BK-D434G mice show hyperexcitability, which likely contributes to the pathogenesis of absence seizures and paroxysmal dyskinesia. A BK channel blocker, paxilline, potently suppresses BK-D434Ginduced hyperexcitability and effectively mitigates absence seizures and locomotor deficits in mice. Our study thus uncovered a neuronal mechanism of BK GOF in absence epilepsy and dyskinesia. Our findings also suggest that BK inhibition is a promising therapeutic strategy for mitigating BK GOF-induced neurological disorders.
Asunto(s)
Canalopatías , Discinesias , Epilepsia Tipo Ausencia , Canales de Potasio de Gran Conductancia Activados por el Calcio , Animales , Discinesias/genética , Epilepsia Tipo Ausencia/tratamiento farmacológico , Epilepsia Tipo Ausencia/genética , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Ratones , Neuronas , ConvulsionesRESUMEN
Large-conductance Ca2+-activated K+ channels (BK channels) are formed by Slo1 subunits as a homotetramer. Besides Ca2+, other divalent cations, such as Cd2+, also activate BK channels when applied intracellularly by shifting the conductance-voltage relation to more negative voltages. However, we found that if the inside-out patch containing BK channels was treated with solution containing reducing agents such as dithiothreitol (DTT), then subsequent Cd2+ application completely inhibited BK currents. The DTT-dependent Cd2+ inhibition could be reversed by treating the patch with solutions containing H2O2, suggesting that a redox reaction regulates the Cd2+ inhibition of BK channels. Similar DTT-dependent Cd2+ inhibition was also observed in a mutant BK channel, Core-MT, in which the cytosolic domain of the channel is deleted, and in the proton-activated Slo3 channels but not observed in the voltage-gated Shaker K+ channels. A possible mechanism for the DTT-dependent Cd2+ inhibition is that DTT treatment breaks one or more disulfide bonds between cysteine pairs in the BK channel protein and the freed thiol groups coordinate with Cd2+ to form an ion bridge that blocks the channel or locks the channel at the closed state. However, surprisingly, none of the mutations of all cysteine residues in Slo1 affect the DTT-dependent Cd2+ inhibition. These results are puzzling, with an apparent contradiction: on one hand, a redox reaction seems to regulate Cd2+ inhibition of the channel, but on the other hand, no cysteine residue in the Slo1 subunit seems to be involved in such inhibition.
Asunto(s)
Cadmio , Ditiotreitol , Oxidación-Reducción , Cadmio/farmacología , Ditiotreitol/farmacología , Animales , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , HumanosRESUMEN
BACKGROUND & AIMS: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of antipruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus. METHODS: Pruritogenicity of lysophosphatidylcholine (LPC), LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. LPC's pruritogenicity involving keratinocyte TRPV4 was studied using genetic and pharmacologic approaches, cultured keratinocytes, ion channel physiology, and structural computational modeling. Activation of pruriceptor sensory neurons by microRNA-146a (miR-146a), secreted from keratinocytes, was identified by in vitro and ex vivo Ca2+ imaging assays. Sera from patients with primary biliary cholangitis were used for measuring the levels of LPC and miR-146a. RESULTS: LPC was robustly pruritic in mice. TRPV4 in skin keratinocytes was essential for LPC-induced itch and itch in mice with cholestasis. Three-dimensional structural modeling, site-directed mutagenesis, and channel function analysis suggested a TRPV4 C-terminal motif for LPC binding and channel activation. In keratinocytes, TRPV4 activation by LPC induced extracellular release of miR-146a, which activated TRPV1+ sensory neurons to cause itch. LPC and miR-146a levels were both elevated in sera of patients with primary biliary cholangitis with itch and correlated with itch intensity. Moreover, LPC and miR-146a were also increased in sera of cholestatic mice and elicited itch in nonhuman primates. CONCLUSIONS: We identified LPC as a novel cholestatic pruritogen that induces itch through epithelia-sensory neuron cross talk, whereby it directly activates skin keratinocyte TRPV4, which rapidly releases miR-146a to activate skin-innervating TRPV1+ pruriceptor sensory neurons. Our findings support the new concept of the skin, as a sensory organ, playing a critical role in cholestatic itch, beyond liver, peripheral sensory neurons, and central neural pathways supporting pruriception.
Asunto(s)
Colestasis/complicaciones , Queratinocitos/metabolismo , Lisofosfatidilcolinas , Prurito/metabolismo , Células Receptoras Sensoriales/metabolismo , Piel/inervación , Canales Catiónicos TRPV/metabolismo , Adulto , Anciano , Animales , Conducta Animal , Células Cultivadas , Colestasis/genética , Colestasis/metabolismo , Colestasis/fisiopatología , Modelos Animales de Enfermedad , Femenino , Humanos , Macaca mulatta , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Prurito/inducido químicamente , Prurito/genética , Prurito/fisiopatología , Transducción de Señal , Canales Catiónicos TRPV/genéticaRESUMEN
TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.
Asunto(s)
Anoctaminas/química , Proteínas de Transferencia de Fosfolípidos/química , Fosfolípidos/química , Animales , Anoctaminas/antagonistas & inhibidores , Anoctaminas/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Ratones , Proteínas de Transferencia de Fosfolípidos/antagonistas & inhibidores , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosfolípidos/metabolismo , Polifenoles/química , Polifenoles/farmacología , Taninos/química , Taninos/farmacologíaRESUMEN
The TMEM16 protein family comprises two novel classes of structurally conserved but functionally distinct membrane transporters that function as Ca2+-dependent Cl- channels (CaCCs) or dual functional Ca2+-dependent ion channels and phospholipid scramblases. Extensive functional and structural studies have advanced our understanding of TMEM16 molecular mechanisms and physiological functions. TMEM16A and TMEM16B CaCCs control transepithelial fluid transport, smooth muscle contraction, and neuronal excitability, whereas TMEM16 phospholipid scramblases mediate the flip-flop of phospholipids across the membrane to allow phosphatidylserine externalization, which is essential in a plethora of important processes such as blood coagulation, bone development, and viral and cell fusion. In this chapter, we summarize the major methods in studying TMEM16 ion channels and scramblases and then focus on the current mechanistic understanding of TMEM16 Ca2+- and voltage-dependent channel gating as well as their ion and phospholipid permeation.
Asunto(s)
Anoctaminas , Proteínas de Transferencia de Fosfolípidos , Anoctaminas/genética , Anoctaminas/metabolismo , Transporte Biológico , Canales de Cloruro/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Proteínas de Transferencia de Fosfolípidos/metabolismo , FosfolípidosRESUMEN
Transmembrane protein 16 (TMEM16) family members play numerous important physiological roles, ranging from controlling membrane excitability and secretion to mediating blood coagulation and viral infection. These diverse functions are largely due to their distinct biophysical properties. Mammalian TMEM16A and TMEM16B are Ca2+-activated Cl- channels (CaCCs), whereas mammalian TMEM16F, fungal afTMEM16, and nhTMEM16 are moonlighting (multifunctional) proteins with both Ca2+-activated phospholipid scramblase (CaPLSase) and Ca2+-activated, nonselective ion channel (CAN) activities. To further understand the biological functions of the enigmatic TMEM16 proteins in different organisms, here, by combining an improved annexin V-based CaPLSase-imaging assay with inside-out patch clamp technique, we thoroughly characterized Subdued, a Drosophila TMEM16 ortholog. We show that Subdued is also a moonlighting transport protein with both CAN and CaPLSase activities. Using a TMEM16F-deficient HEK293T cell line to avoid strong interference from endogenous CaPLSases, our functional characterization and mutagenesis studies revealed that Subdued is a bona fide CaPLSase. Our finding that Subdued is a moonlighting TMEM16 expands our understanding of the molecular mechanisms of TMEM16 proteins and their evolution and physiology in both Drosophila and humans.
Asunto(s)
Anoctaminas/metabolismo , Calcio/metabolismo , Proteínas de Drosophila/metabolismo , Fosfolípidos/metabolismo , Animales , Anoctaminas/genética , Transporte Biológico , Cationes , Drosophila , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Transporte Iónico , Permeabilidad , Proteínas de Transferencia de Fosfolípidos/metabolismoRESUMEN
BACKGROUND: The mutations of KCNMA1 BK-type K+ channel have been identified in patients with various movement disorders. The underlying pathophysiology and corresponding therapeutics are lacking. OBJECTIVES: To report our clinical and biophysical characterizations of a novel de novo KCNMA1 variant, as well as an effective therapy for the patient's dystonia-atonia spells. METHODS: Combination of phenotypic characterization, therapy, and biophysical characterization of the patient and her mutation. RESULTS: The patient had >100 dystonia-atonia spells per day with mild cerebellar atrophy. She also had autism spectrum disorder, intellectual disability, and attention deficit hyperactivity disorder. Whole-exome sequencing identified a heterozygous de novo BK N536H mutation. Our biophysical characterization demonstrates that N536H is a gain-of-function mutation with markedly enhanced voltage-dependent activation. Remarkably, administration of dextroamphetamine completely suppressed the dystonia-atonia spells. CONCLUSIONS: BK N536H is a gain-of-function that causes dystonia and other neurological symptoms. Our stimulant therapy opens a new avenue to mitigate KCNMA1-linked movement disorders. © 2020 International Parkinson and Movement Disorder Society.
Asunto(s)
Trastorno del Espectro Autista , Distonía , Discapacidad Intelectual , Distonía/tratamiento farmacológico , Distonía/genética , Femenino , Mutación con Ganancia de Función , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Mutación/genéticaRESUMEN
Coupling between the activation gate and sensors of physiological stimuli during ion channel activation is an important, but not well-understood, molecular process. One difficulty in studying sensor-gate coupling is to distinguish whether a structural perturbation alters the function of the sensor, the gate, or their coupling. BK channels are activated by membrane voltage and intracellular Ca(2+) via allosteric mechanisms with coupling among the activation gate and sensors quantitatively defined, providing an excellent model system for studying sensor-gate coupling. By studying BK channels expressed in Xenopus oocytes, here we show that mutation E219R in S4 alters channel function by two independent mechanisms: one is to change voltage sensor activation, shifting voltage dependence, and increase valence of gating charge movements; the other is to regulate coupling among the activation gate, voltage sensor, and Ca(2+) binding via electrostatic interactions with E321/E324 located in the cytosolic side of S6 in a neighboring subunit, resulting in a shift of the voltage dependence of channel opening and increased Ca(2+) sensitivity. These results suggest a structural arrangement of the inner pore of BK channels differing from that in other voltage gated channels.
Asunto(s)
Calcio/química , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Aminoácidos/química , Animales , Células Cultivadas , Electricidad Estática , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.
Asunto(s)
Canales de Cloruro/metabolismo , Mucinas/metabolismo , Contracción Muscular/fisiología , Músculo Liso/fisiología , Sistema Respiratorio/citología , Sistema Respiratorio/metabolismo , Animales , Anoctamina-1 , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Ratones , Microscopía FluorescenteRESUMEN
Large-conductance, voltage-, and Ca²âº-dependent K⺠(BK) channels are broadly expressed in various tissues to modulate neuronal activity, smooth muscle contraction, and secretion. BK channel activation depends on the interactions among the voltage sensing domain (VSD), the cytosolic domain (CTD), and the pore gate domain (PGD) of the Slo1 α-subunit, and is further regulated by accessory ß subunits (ß1-ß4). How ß subunits fine-tune BK channel activation is critical to understand the tissue-specific functions of BK channels. Multiple sites in both Slo1 and the ß subunits have been identified to contribute to the interaction between Slo1 and the ß subunits. However, it is unclear whether and how the interdomain interactions among the VSD, CTD, and PGD are altered by the ß subunits to affect channel activation. Here we show that human ß1 and ß2 subunits alter interactions between bound Mg²âº and gating charge R213 and disrupt the disulfide bond formation at the VSD-CTD interface of mouse Slo1, indicating that the ß subunits alter the VSD-CTD interface. Reciprocally, mutations in the Slo1 that alter the VSD-CTD interaction can specifically change the effects of the ß1 subunit on the Ca²âº activation and of the ß2 subunit on the voltage activation. Together, our data suggest a novel mechanism by which the ß subunits modulated BK channel activation such that a ß subunit may interact with the VSD or the CTD and alter the VSD-CTD interface of the Slo1, which enables the ß subunit to have effects broadly on both voltage and Ca²âº-dependent activation.
Asunto(s)
Membrana Celular/metabolismo , Citosol/metabolismo , Activación del Canal Iónico/fisiología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Animales , Membrana Celular/química , Citosol/química , Femenino , Humanos , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Ratones , Estructura Terciaria de Proteína , Xenopus laevisRESUMEN
The large conductance calcium-sensitive potassium (BK) channel is widely expressed in many organs and tissues, but its in vivo physiological functions have not been fully defined. Here we report a genetic locus associated with a human syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia on chromosome 10q22 and show that a mutation of the alpha subunit of the BK channel causes this syndrome. The mutant BK channel had a markedly greater macroscopic current. Single-channel recordings showed an increase in open-channel probability due to a three- to fivefold increase in Ca(2+) sensitivity. We propose that enhancement of BK channels in vivo leads to increased excitability by inducing rapid repolarization of action potentials, resulting in generalized epilepsy and paroxysmal dyskinesia by allowing neurons to fire at a faster rate. These results identify a gene that is mutated in generalized epilepsy and paroxysmal dyskinesia and have implications for the pathogenesis of human epilepsy, the neurophysiology of paroxysmal movement disorders and the role of BK channels in neurological disease.
Asunto(s)
Corea/genética , Epilepsia Generalizada/genética , Canales de Potasio Calcio-Activados/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Preescolar , Corea/complicaciones , Cromosomas Humanos Par 10 , Secuencia Conservada , Cricetinae , Cricetulus , Epilepsia Generalizada/complicaciones , Femenino , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Datos de Secuencia Molecular , Mutación , Oocitos/fisiología , Linaje , Canales de Potasio Calcio-Activados/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/fisiología , Xenopus laevisRESUMEN
The calcium-activated TMEM16 proteins and the mechanosensitive/osmolarity-activated OSCA/TMEM63 proteins belong to the Transmembrane Channel/Scramblase (TCS) superfamily. Within the superfamily, OSCA/TMEM63 proteins, as well as TMEM16A and TMEM16B, likely function solely as ion channels. However, the remaining TMEM16 members, including TMEM16F, maintain an additional function as scramblases, rapidly exchanging phospholipids between leaflets of the membrane. Although recent studies have advanced our understanding of TCS structure-function relationships, the molecular determinants of TCS ion and lipid permeation remain unclear. Here we show that single lysine mutations in transmembrane helix (TM) 4 allow non-scrambling TCS members to permeate phospholipids. This study highlights the key role of TM 4 in controlling TCS ion and lipid permeation and offers novel insights into the evolution of the TCS superfamily, suggesting that, like TMEM16s, the OSCA/TMEM63 family maintains a conserved potential to permeate ions and phospholipids.
RESUMEN
The TMEM16A calcium-activated chloride channel is a promising therapeutic target for various diseases. Niclosamide, an anthelmintic medication, has been considered a TMEM16A inhibitor for treating asthma and chronic obstructive pulmonary disease (COPD) but was recently found to possess broad-spectrum off-target effects. Here, we show that, under physiological Ca2+ (200-500 nM) and voltages, niclosamide acutely potentiates TMEM16A. Our computational and functional characterizations pinpoint a putative niclosamide binding site on the extracellular side of TMEM16A. Mutations in this site attenuate the potentiation. Moreover, niclosamide potentiates endogenous TMEM16A in vascular smooth muscle cells, triggers intracellular calcium increase, and constricts the murine mesenteric artery. Our findings advise caution when considering clinical applications of niclosamide as a TMEM16A inhibitor. The identification of the putative niclosamide binding site provides insights into the mechanism of TMEM16A pharmacological modulation and provides insights into developing specific TMEM16A modulators to treat human diseases.
Asunto(s)
Anoctamina-1 , Niclosamida , Vasoconstricción , Niclosamida/farmacología , Anoctamina-1/metabolismo , Anoctamina-1/genética , Animales , Ratones , Humanos , Vasoconstricción/efectos de los fármacos , Células HEK293 , Sitios de Unión , Calcio/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , MasculinoRESUMEN
We investigated the hemotoxic effects of three North American pit vipers in healthy human donor blood. Using experiments focusing on platelet and red blood cell activity, we found differential effects of these venoms on these cellular components. Platelet aggregation was most induced by C. adamanteus. Platelet activation was highest with C. atrox. Red blood cells had calcium expression and erythrocyte formation most induced by C. adamanteus and A. piscivorus. These results demonstrate the complex interplay of individual cellular effects with clinical presentations seen in envenomings from these species.
Asunto(s)
Plaquetas , Eritrocitos , Humanos , Eritrocitos/efectos de los fármacos , Plaquetas/efectos de los fármacos , Animales , Venenos de Crotálidos/toxicidad , Agregación Plaquetaria/efectos de los fármacos , Calcio/metabolismo , Calcio/sangre , Activación Plaquetaria/efectos de los fármacos , CrotalinaeRESUMEN
Human trophoblast organoids (TOs) are a three-dimensional ex vivo culture model that can be used to study various aspects of placental development, physiology, and pathology. Previously, we showed that TOs derived from full-term human placental tissue could be used as models of trophoblast innate immune signaling and teratogenic virus infections. Here, we developed a method to culture TOs under conditions that recapitulate the cellular orientation of chorionic villi in vivo , with the multi-nucleated syncytiotrophoblast (STB) localized to the outer surface of organoids and the proliferative cytotrophoblasts (CTBs) located on the inner surface. We show that standard TOs containing the STB layer inside the organoid (STB in ) develop into organoids containing the STB on the outer surface (STB out ) when cultured in suspension with gentle agitation. STB out organoids secrete higher levels of select STB-associated hormones and cytokines, including human chorionic gonadotropin (hCG) and interferon (IFN)-λ2. Using membrane capacitance measurements, we also show that the outermost surface of STB out organoids contain large syncytia comprised of >50 nuclei compared to STB in organoids that contain small syncytia (<10 nuclei) and mononuclear cells. The growth of TOs under conditions that mimic the cellular orientation of chorionic villi in vivo thus allows for the study of a variety of aspects of placental biology under physiological conditions.