RESUMEN
Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.
Asunto(s)
Canales Iónicos , Animales , Humanos , Ratones , Secuencia de Aminoácidos , Células HEK293 , Canales Iónicos/metabolismo , Canales Iónicos/química , Membrana Dobles de Lípidos/metabolismo , Mecanotransducción Celular , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Mutación , Subunidades de Proteína/metabolismoRESUMEN
Malformation during cortical development can disrupt the balance of excitatory and inhibitory neural circuits, contributing to various psychiatric and developmental disorders. One of the critical factors of cortical neural networks is the fine regulation of neurogenesis through mechanical cues, such as shear stress and substrate stiffness. Piezo1, a mechanically-activated channel, serves as a transducer for these mechanical cues, regulating embryogenesis. However, specific cell-type expression patterns of this channel during cortical development have not yet been characterized. In the present study, we conducted an RNAscope experiment to visualize the location of Piezo1 transcripts with embryonic neuronal/glial lineage cell markers. Our analysis covered coronal sections of the mouse forebrain on embryonic day 12.5 (E12.5), E14.5, E16.5, and E18.5. In addition, applying Yoda1, a specific Piezo1 agonist, evoked distinct calcium elevation in piriform cortices of E16.5 and E18.5 embryonic slices. Furthermore, pharmacological activation or inhibition of this channel significantly modulated the migration of neurosphere-derived cells in vitro. These findings contribute valuable insights to the field of mechanobiology and provide an understanding of the intricate processes underlying embryonic brain development.
Asunto(s)
Canales Iónicos , Neurogénesis , Animales , Ratones , Canales Iónicos/genética , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Neurogénesis/genética , Prosencéfalo/metabolismoRESUMEN
Anisotropically organized neural networks are indispensable routes for functional connectivity in the brain, which remains largely unknown. While prevailing animal models require additional preparation and stimulation-applying devices and have exhibited limited capabilities regarding localized stimulation, no in vitro platform exists that permits spatiotemporal control of chemo-stimulation in anisotropic three-dimensional (3D) neural networks. We present the integration of microchannels seamlessly into a fibril-aligned 3D scaffold by adapting a single fabrication principle. We investigated the underlying physics of elastic microchannels' ridges and interfacial sol-gel transition of collagen under compression to determine a critical window of geometry and strain. We demonstrated the spatiotemporally resolved neuromodulation in an aligned 3D neural network by local deliveries of KCl and Ca2+ signal inhibitors, such as tetrodotoxin, nifedipine, and mibefradil, and also visualized Ca2+ signal propagation with a speed of ~3.7 µm/s. We anticipate that our technology will pave the way to elucidate functional connectivity and neurological diseases associated with transsynaptic propagation.
Asunto(s)
Encéfalo , Colágeno , Animales , Encéfalo/fisiologíaRESUMEN
Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9-deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Thus, we concluded that the signal amplification by ANO9 is important for mammalian olfactory transduction.
Asunto(s)
Neuronas Receptoras Olfatorias , Olfato , Animales , Ratones , Anoctaminas/metabolismo , Mamíferos/metabolismo , Odorantes , Bulbo Olfatorio/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Olfato/fisiologíaRESUMEN
Several studies have documented the broad-spectrum bioactivities of a lotus seed (Plumula nelumbinis [PN]) green embryo extract. However, the specific bioactive components and associated molecular mechanisms remain largely unknown. This study aimed to identify the ion channel-activating mechanisms of PN extracts. Using fluorometric imaging and patch-clamp recordings, PN extracts were screened for calcium channel activation in dorsal root ganglion (DRG) neurons. The TRPV1 channels in DRG neurons were strongly activated by the PN extract (mean amplitude of 131 ± 45 pA at 200 µg/mL) and its purified glycosyloxyflavone narcissoside (401 ± 271 pA at 100 µM). Serial treatment with a 200 µg/mL PN extract in TRPV1-overexpressing HEK293T cells induced robust desensitization to 10 ± 10% of the initial current amplitude. Thus, we propose that the PN extract and narcissoside function as TRPV1 agonists. This new finding may advance our knowledge regarding the traditional and scientific functions of PN in human health and disease.
Asunto(s)
Ganglios Espinales , Extractos Vegetales , Canales Catiónicos TRPV , Calcio/metabolismo , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Lotus/química , Extractos Vegetales/farmacología , Semillas/química , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/genéticaRESUMEN
Glucose homeostasis is initially regulated by the pancreatic hormone insulin. Glucose-stimulated insulin secretion in ß-cells is composed of two cellular mechanisms: a high glucose concentration not only depolarizes the membrane potential of the ß-cells by ATP-sensitive K+ channels but also induces cell inflation, which is sufficient to release insulin granules. However, the molecular identity of the stretch-activated cation channel responsible for the latter pathway remains unknown. Here, we demonstrate that Tentonin 3/TMEM150C (TTN3), a mechanosensitive channel, contributes to glucose-stimulated insulin secretion by mediating cation influx. TTN3 is expressed specifically in ß-cells and mediates cation currents to glucose and hypotonic stimulations. The glucose-induced depolarization, firing activity, and Ca2+ influx of ß-cells were significantly lower in Ttn3-/- mice. More importantly, Ttn3-/- mice show impaired glucose tolerance with decreased insulin secretion in vivo. We propose that TTN3, as a stretch-activated cation channel, contributes to glucose-stimulated insulin secretion.
Asunto(s)
Calcio/metabolismo , Intolerancia a la Glucosa/patología , Glucosa/farmacología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Proteínas de la Membrana/fisiología , Animales , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Edulcorantes/farmacologíaRESUMEN
The baroreceptor reflex is a powerful neural feedback that regulates arterial pressure (AP). Mechanosensitive channels transduce pulsatile AP to electrical signals in baroreceptors. Here we show that tentonin 3 (TTN3/TMEM150C), a cation channel activated by mechanical strokes, is essential for detecting AP changes in the aortic arch. TTN3 was expressed in nerve terminals in the aortic arch and nodose ganglion (NG) neurons. Genetic ablation of Ttn3 induced ambient hypertension, tachycardia, AP fluctuations, and impaired baroreflex sensitivity. Chemogenetic silencing or activation of Ttn3+ neurons in the NG resulted in an increase in AP and heart rate, or vice versa. More important, overexpression of Ttn3 in the NG of Ttn3-/- mice reversed the cardiovascular changes observed in Ttn3-/- mice. We conclude that TTN3 is a molecular component contributing to the sensing of dynamic AP changes in baroreceptors.
Asunto(s)
Aorta Torácica , Presión Sanguínea , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Ganglio Nudoso , Presorreceptores , Animales , Aorta Torácica/inervación , Aorta Torácica/metabolismo , Aorta Torácica/fisiopatología , Células HEK293 , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ganglio Nudoso/metabolismo , Ganglio Nudoso/fisiopatología , Presorreceptores/metabolismo , Presorreceptores/fisiopatología , Taquicardia/genética , Taquicardia/metabolismo , Taquicardia/fisiopatologíaRESUMEN
Low-intensity, low-frequency ultrasound (LILFU) is the next-generation, non-invasive brain stimulation technology for treating various neurological and psychiatric disorders. However, the underlying cellular and molecular mechanism of LILFU-induced neuromodulation has remained unknown. Here, we report that LILFU-induced neuromodulation is initiated by opening of TRPA1 channels in astrocytes. The Ca2+ entry through TRPA1 causes a release of gliotransmitters including glutamate through Best1 channels in astrocytes. The released glutamate activates NMDA receptors in neighboring neurons to elicit action potential firing. Our results reveal an unprecedented mechanism of LILFU-induced neuromodulation, involving TRPA1 as a unique sensor for LILFU and glutamate-releasing Best1 as a mediator of glia-neuron interaction. These discoveries should prove to be useful for optimization of human brain stimulation and ultrasonogenetic manipulations of TRPA1.
Asunto(s)
Astrocitos/metabolismo , Ácido Glutámico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Canal Catiónico TRPA1/genética , Ultrasonografía , Animales , Masculino , Ratones , Distribución Aleatoria , Canal Catiónico TRPA1/metabolismoRESUMEN
Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1-deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1-deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1-deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl- secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis.
Asunto(s)
Anoctamina-1/fisiología , Carbacol/farmacología , Cloruros/metabolismo , Toxina del Cólera/farmacología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Animales , Anoctamina-1/genética , Calcio/metabolismo , Canales de Cloruro/genética , Canales de Cloruro/fisiología , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colitis/patología , Femenino , Intestinos/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Vías Secretoras/efectos de los fármacos , Vías Secretoras/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Neural stem cells (NSCs) are primary progenitor cells in the early developmental stage in the brain that initiate a diverse lineage of differentiated neurons and glia. Radial glial cells (RGCs), a type of neural stem cell in the ventricular zone, are essential for nurturing and delivering new immature neurons to the appropriate cortical target layers. Here we report that Anoctamin 1 (ANO1)/TMEM16A, a Ca2+-activated chloride channel, mediates the Ca2+-dependent process extension of RGCs. ANO1 is highly expressed and functionally active in RGCs of the mouse embryonic ventricular zone. Knockdown of ANO1 suppresses RGC process extension and protrusions, whereas ANO1 overexpression stimulates process extension. Among various trophic factors, brain-derived neurotrophic factor (BDNF) activates ANO1, which is required for BDNF-induced process extension in RGCs. More importantly, Ano1-deficient mice exhibited disrupted cortical layers and reduced cortical thickness. We thus conclude that the regulation of RGC process extension by ANO1 contributes to the normal formation of mouse embryonic brain.
Asunto(s)
Anoctamina-1/fisiología , Encéfalo/citología , Encéfalo/embriología , Neuroglía/citología , Animales , Anoctamina-1/genética , Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Cloruros/metabolismo , Regulación hacia Abajo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroglía/metabolismo , Regulación hacia ArribaRESUMEN
Mechanosensation is essential for various physiological processes, and it is mediated by mechanotransduction channels. Recently, we reported that TMEM150C/Tentonin 3 (TTN3) confers mechanically activated currents with slow inactivation kinetics in several cell types, including dorsal root ganglion neurons (Hong et al., 2016). The accompanying Matters Arising by Dubin, Murthy, and colleagues confirms that naive heterologous cells demonstrate a mechanically activated current, but finds that this response is absent in CRISPR-Cas9 Piezo1 knockout cell lines and suggests that TTN3 is a modulator of Piezo1. We present and discuss evidence based on co-expression of TTN3 and Peizo1 and mutant variants of the pore region of TTN3 to support that TTN3 is a pore-forming unit, not an amplifying adaptor for Piezo1 activity. This Matters Arising Response paper, along with Zhao et al. (2017), addresses the Matters Arising from Dubin et al. (2017), published concurrently in this issue of Neuron.
Asunto(s)
Ganglios Espinales/citología , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Neuronas/fisiología , Transporte Biológico , Línea Celular , HumanosRESUMEN
Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C is activated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The MA TTN3 currents were inhibited by known blockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinated movements and abnormal gait. Thus, TTN3 appears to be a component of a mechanosensitive channel with a slow inactivation rate and contributes to motor coordination. Identification of this gene advances our understanding of the various types of mechanosensations, including proprioception.
Asunto(s)
Ganglios Espinales/metabolismo , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Mecanorreceptores/fisiología , Ratones Transgénicos , Tacto/fisiologíaRESUMEN
BACKGROUND: Korean Red Ginseng-a steamed root of Panax ginseng Meyer-has long been used as a traditional medicine in Asian countries. Its antipruritic effect was recently found, but no molecular mechanisms were revealed. Thus, the current study focused on determining the underlying molecular mechanism of Korean Red Ginseng extract (RGE) against histamine-induced itch at the peripheral sensory neuronal level. METHODS: To examine the antipruritic effect of RGE, we performed in vivo scratching behavior test in mice, as well as in vitro calcium imaging and whole-cell patch clamp experiments to elucidate underlying molecular mechanisms. RESULTS: The results of our in vivo study confirmed that RGE indeed has an antipruritic effect on histamine-induced scratching in mice. In addition, RGE showed a significant inhibitory effect on histamine-induced responses in primary cultures of mouse dorsal root ganglia, suggesting that RGE has a direct inhibitory effect on sensory neuronal level. Results of further experiments showed that RGE inhibits histamine-induced responses on cells expressing both histamine receptor subtype 1 and TRPV1 ion channel, indicating that RGE blocks the histamine receptor type 1/TRPV1 pathway in sensory neurons, which is responsible for histamine-dependent itch sensation. CONCLUSION: The current study found for the first time that RGE effectively blocks histamine-induced itch in peripheral sensory neurons. We believe that the current results will provide an insight on itch transmission and will be helpful in understanding how RGE exerts its antipruritic effects.
RESUMEN
Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate, causing lower urinary tract symptoms in elderly men worldwide. However, the molecular mechanism underlying the pathogenesis of BPH is unclear. Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride channel (CaCC) that mediates various physiological functions. Here, we demonstrate that it is essential for testosterone-induced BPH. ANO1 was highly amplified in dihydrotestosterone (DHT)-treated prostate epithelial cells, whereas the selective knockdown of ANO1 inhibited DHT-induced cell proliferation. Three androgen-response elements were found in the ANO1 promoter region, which is relevant for the DHT-dependent induction of ANO1. Administration of the ANO1 blocker or Ano1 small interfering RNA, inhibited prostate enlargement and reduced histological abnormalities in vivo. We therefore concluded that ANO1 is essential for the development of prostate hyperplasia and is a potential target for the treatment of BPH.
Asunto(s)
Canales de Cloruro/metabolismo , Proteínas de Neoplasias/metabolismo , Próstata/metabolismo , Próstata/patología , Testosterona/farmacología , Animales , Anoctamina-1 , Calcio/farmacología , Canales de Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Dihidrotestosterona/farmacología , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Hiperplasia , Inyecciones , Activación del Canal Iónico/efectos de los fármacos , Luciferasas/metabolismo , Masculino , Regiones Promotoras Genéticas/genética , Próstata/efectos de los fármacos , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , ARN Interferente Pequeño/metabolismo , Ratas Wistar , Elementos de Respuesta/genética , Taninos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Poncirus fructus (PF), also known as a dried immature fruit of Poncirus trifoliata (L.) Raf. (Rutaceae), has long been traditionally used for the various gastrointestinal disorders in Eastern Asia. AIM OF STUDY: The aqueous extract of PF (PF-W) has the strong prokinetic effect, yet the underlying mechanism is still elusive. The present study investigated whether PF-W has any effect on motilin receptor or ghrelin receptor, since these receptors enhance intestinal motility when activated. MATERIALS AND METHODS: The effect of PF-W and its components on motilin or ghrelin receptor was determined by calcium imaging and whole-cell patch clamp methods. RESULTS: PF-W activates the ghrelin receptor, but not the motilin receptor, resulting in a transient increase of intracellular calcium levels. Furthermore, among various constituents of PF, only naringin and naringenin evoked the intracellular calcium augmentation via the ghrelin receptor. Moreover, cortistatin-8 - a ghrelin receptor inhibitor - specifically blocked naringin- and naringenin-induced calcium increases. In addition, naringin and naringenin induced inward currents in ghrelin receptor-expressing cells under whole-cell patch clamp configuration. CONCLUSION: PF-W activates the ghrelin receptor, and naringin and naringenin are key constituents responsible for the activation of ghrelin receptor. Therefore, the present study suggests that the ghrelin receptor is a molecular entity responsible for the strong prokinetic activity of PF-W.