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In a recent study, Liang, Wilson, and colleagues demonstrated that the H+-selective ion channel OTOP1, responsible for sour taste transduction, also functions as a gustatory sensor for ammonium in mice. Additionally, this research revealed a novel mode of channel activation by intracellular alkalinization, which is conserved across vertebrate species.
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Compuestos de Amonio , Animales , Ratones , Canales IónicosRESUMEN
Nonalcoholic steatohepatitis (NASH) is a progressive disorder with aberrant lipid accumulation and subsequent inflammatory and profibrotic response. Therapeutic efforts at lipid reduction via increasing cytoplasmic lipolysis unfortunately worsens hepatitis due to toxicity of liberated fatty acid. An alternative approach could be lipid reduction through autophagic disposal, i.e., lipophagy. We engineered a synthetic adaptor protein to induce lipophagy, combining a lipid droplet-targeting signal with optimized LC3-interacting domain. Activating hepatocyte lipophagy in vivo strongly mitigated both steatosis and hepatitis in a diet-induced mouse NASH model. Mechanistically, activated lipophagy promoted the excretion of lipid from hepatocytes, thereby suppressing harmful intracellular accumulation of nonesterified fatty acid. A high-content compound screen identified alpelisib and digoxin, clinically-approved compounds, as effective activators of lipophagy. Administration of alpelisib or digoxin in vivo strongly inhibited the transition to steatohepatitis. These data thus identify lipophagy as a promising therapeutic approach to prevent NASH progression.
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Hepatitis , Enfermedad del Hígado Graso no Alcohólico , Animales , Ratones , Autofagia , Digoxina/farmacología , Ácidos Grasos/metabolismo , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Lípidos , Hígado/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
Salt taste, the taste of sodium chloride (NaCl), is mechanistically one of the most complex and puzzling among basic tastes. Sodium has essential functions in the body but causes harm in excess. Thus, animals use salt taste to ingest the right amount of salt, which fluctuates by physiological needs: typically, attraction to low salt concentrations and rejection of high salt. This concentration-valence relationship is universally observed in terrestrial animals, and research has revealed complex peripheral codes for NaCl involving multiple taste pathways of opposing valence. Sodium-dependent and -independent pathways mediate attraction and aversion to NaCl, respectively. Gustatory sensors and cells that transduce NaCl have been uncovered, along with downstream signal transduction and neurotransmission mechanisms. However, much remains unknown. This article reviews classical and recent advances in our understanding of the molecular and cellular mechanisms underlying salt taste in mammals and insects and discusses perspectives on human salt taste.
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Papilas Gustativas , Gusto , Animales , Humanos , Gusto/fisiología , Cloruro de Sodio/metabolismo , Papilas Gustativas/metabolismo , Sodio/metabolismo , Transducción de Señal , Mamíferos/metabolismoRESUMEN
Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.
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Relojes Circadianos , Proteínas de Unión al GTP Monoméricas , Animales , Canales de Calcio Tipo L/metabolismo , Ritmo Circadiano/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP Monoméricas/metabolismo , Núcleo Supraquiasmático/metabolismoRESUMEN
Among calcium homeostasis modulator (CALHM) family members, CALHM1 and 3 together form a voltage-gated large-pore ion channel called CALHM1/3. CALHM1/3 plays an essential role in taste perception by mediating neurotransmitter release at channel synapses of taste bud cells. However, it is poorly understood how CALHM1/3 is regulated. Biochemical analyses of the two subunits following site-directed mutagenesis and pharmacological treatments established that both CALHM1 and 3 were N-glycosylated at single Asn residues in their second extracellular loops. Biochemical and electrophysiological studies revealed that N-glycan acquisition on CALHM1 and 3, respectively, controls the biosynthesis and gating kinetics of the CALHM1/3 channel. Furthermore, failure in subsequent remodeling of N-glycans decelerated the gating kinetics. Thus, the acquisition of N-glycans on both subunits and their remodeling differentially contribute to the functional expression of CALHM1/3. Meanwhile, metabolic labeling and acyl-biotin exchange assays combined with genetic modification demonstrated that CALHM3 was reversibly palmitoylated at three intracellular Cys residues. Screening of the DHHC protein acyltransferases identified DHHC3 and 15 as CALHM3 palmitoylating enzymes. The palmitoylation-deficient mutant CALHM3 showed a normal degradation rate and interaction with CALHM1. However, the same mutation markedly attenuated the channel activity but not surface localization of CALHM1/3, suggesting that CALHM3 palmitoylation is a critical determinant of CALHM1/3 activity but not its formation or forward trafficking. Overall, this study characterized N-glycosylation and S-palmitoylation of CALHM1/3 subunits and clarified their differential contributions to its functional expression, providing insights into the fine control of the CALHM1/3 channel and associated physiological processes.
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Canales de Calcio/metabolismo , Activación del Canal Iónico , Lipoilación , Glicoproteínas de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Canales de Calcio/química , Glicosilación , Células HeLa , Humanos , Glicoproteínas de Membrana/químicaRESUMEN
The variety of taste sensations, including sweet, umami, bitter, sour, and salty, arises from diverse taste cells, each of which expresses specific taste sensor molecules and associated components for downstream signal transduction cascades. Recent years have witnessed major advances in our understanding of the molecular mechanisms underlying transduction of basic tastes in taste buds, including the identification of the bona fide sour sensor H+ channel OTOP1, and elucidation of transduction of the amiloride-sensitive component of salty taste (the taste of sodium) and the TAS1R-independent component of sweet taste (the taste of sugar). Studies have also discovered an unconventional chemical synapse termed "channel synapse" which employs an action potential-activated CALHM1/3 ion channel instead of exocytosis of synaptic vesicles as the conduit for neurotransmitter release that links taste cells to afferent neurons. New images of the channel synapse and determinations of the structures of CALHM channels have provided structural and functional insights into this unique synapse. In this review, we discuss the current view of taste transduction and neurotransmission with emphasis on recent advances in the field.
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Sinapsis/clasificación , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Papilas Gustativas/fisiología , Gusto/fisiología , Animales , HumanosRESUMEN
Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated adenosine triphosphate (ATP)-release channels involved in neural functions including neurotransmission in gustation. Here, we present the cryo-electron microscopy (EM) structures of killifish CALHM1, human CALHM2, and Caenorhabditis elegans CLHM-1 at resolutions of 2.66, 3.4, and 3.6 Å, respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonamer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the intersubunit interactions at the transmembrane domain (TMD) and the TMD-intracellular domain linker define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels.
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Sodium taste regulates salt intake. The amiloride-sensitive epithelial sodium channel (ENaC) is the Na+ sensor in taste cells mediating attraction to sodium salts. However, cells and intracellular signaling underlying sodium taste in taste buds remain long-standing enigmas. Here, we show that a subset of taste cells with ENaC activity fire action potentials in response to ENaC-mediated Na+ influx without changing the intracellular Ca2+ concentration and form a channel synapse with afferent neurons involving the voltage-gated neurotransmitter-release channel composed of calcium homeostasis modulator 1 (CALHM1) and CALHM3 (CALHM1/3). Genetic elimination of ENaC in CALHM1-expressing cells as well as global CALHM3 deletion abolished amiloride-sensitive neural responses and attenuated behavioral attraction to NaCl. Together, sodium taste is mediated by cells expressing ENaC and CALHM1/3, where oral Na+ entry elicits suprathreshold depolarization for action potentials driving voltage-dependent neurotransmission via the channel synapse. Thus, all steps in sodium taste signaling are voltage driven and independent of Ca2+ signals. This work also reveals ENaC-independent salt attraction.
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Potenciales de Acción/fisiología , Calcio/metabolismo , Canales Epiteliales de Sodio/metabolismo , Sodio/metabolismo , Papilas Gustativas/citología , Gusto/fisiología , Potenciales de Acción/efectos de los fármacos , Amilorida/farmacología , Animales , Canales de Calcio/metabolismo , Células Quimiorreceptoras/metabolismo , Células Quimiorreceptoras/fisiología , Bloqueadores del Canal de Sodio Epitelial/farmacología , Ratones , Neuronas Aferentes/metabolismo , Técnicas de Placa-Clamp , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica , Papilas Gustativas/metabolismo , Papilas Gustativas/fisiologíaRESUMEN
The CALHM1/CALHM3 channel in the basolateral membrane of polarized taste cells mediates neurotransmitter release. However, mechanisms regulating its localization remain unexplored. Here, we identified CALHM1/CALHM3 in the basolateral membrane of type II taste cells in discrete puncta localized close to afferent nerve fibers. As in taste cells, CALHM1/CALHM3 was present in the basolateral membrane of model epithelia, although it was distributed throughout the membrane and did not show accumulation in puncta. We identified canonical basolateral sorting signals in CALHM1 and CALHM3: tyrosine-based and dileucine motifs. However, basolateral sorting remained intact in mutated channels lacking those signals, suggesting that non-canonical signals reside elsewhere. Our study demonstrates intrinsic basolateral sorting of CALHM channels in polarized cells, and provides mechanistic insights.
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Canales de Calcio/metabolismo , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Papilas Gustativas/metabolismo , Secuencia de Aminoácidos , Animales , Canales de Calcio/genética , Línea Celular Tumoral , Polaridad Celular/genética , Células Cultivadas , Perros , Humanos , Activación del Canal Iónico/genética , Células de Riñón Canino Madin Darby , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Transmisión Sináptica/genética , Papilas Gustativas/citologíaRESUMEN
Taste sensation is initiated in sensory cells within the taste buds (taste cells), in which the cooperation of many signaling molecules leads to the coding and transmission of information on the quality and intensity of taste to the afferent gustatory nerves. Here, we describe our method for inducing foreign gene expression in taste cells of fungiform papillae in a living mouse using a recombinant adeno-associated virus (AAV) vector, enabling us to study and control the function of a gene product in vivo. Among the serotypes tested to date, only AAV-DJ, a synthetic serotype, can transduce taste cells in vivo. We also describe how to validate intragemmal foreign gene expression in fungiform taste buds using an immunohistochemical approach.
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Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Papilas Gustativas/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes Reporteros , Inmunohistoquímica , Masculino , Ratones , TransgenesRESUMEN
BACKGROUND/AIMS: Epithelial Na+ channels (ENaCs) play crucial roles in control of blood pressure by determining the total amount of renal Na+ reabsorption, which is regulated by various factors such as aldosterone, vasopressin, insulin and osmolality. The intracellular trafficking process of ENaCs regulates the amount of the ENaC-mediated Na+ reabsorption in the collecting duct of the kidney mainly by determining the number of ENaC expressed at the apical membrane of epithelial cells. Although we previously reported protein tyrosine kinases (PTKs) contributed to the ENaC-mediated epithelial Na+ reabsorption, we have no information on the role of PTKs in the intracellular ENaC trafficking. METHODS: Using the mathematical model recently established in our laboratory, we studied the effect of PTKs inhibitors (PTKIs), AG1296 (10 µM: an inhibitor of the PDGF receptor (PDGFR)) and AG1478 (10 µM: an inhibitor of the EGF receptor (EGFR)) on the rates of the intracellular ENaC trafficking in renal epithelial A6 cells endogenously expressing ENaCs. RESULTS: We found that application of PTKIs significantly reduced the insertion rate of ENaC to the apical membrane by 56%, the recycling rate of ENaC by 83%, the cumulative time of an individual ENaC staying in the apical membrane by 27%, the whole life-time after the first insertion of ENaC by 47%, and the cumulative Na+ absorption by 61%, while the degradation rate was increased to 3.8-fold by application of PTKIs. These observations indicate that PTKs contribute to the processes of insertion, recycling and degradation of ENaC in the intracellular trafficking process under a hypotonic condition. CONCLUSION: The present study indicates that application of EGFR and PDGFR-inhibitable PTKIs reduced the insertion rate (kI), and the recycling rate (kR) of ENaCs, but increased degradation rate (kD) in renal A6 epithelial cells under a hypotonic condition. These observations indicate that hypotonicity increases the surface expression of ENaCs by increasing the insertion rate (kI) and the recycling rate (kR) of ENaCs associated with a decrease in the degradation rate but without any significant effects on the endocytotic rate (kE) in EGFR and PDGFR-related PTKs-mediated pathways.
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Canales Epiteliales de Sodio/metabolismo , Modelos Teóricos , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas/efectos de los fármacos , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Línea Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Soluciones Hipotónicas/química , Soluciones Hipotónicas/farmacología , Riñón/citología , Cinética , Quinazolinas/farmacología , Sodio/metabolismo , Tirfostinos/farmacología , Xenopus laevisRESUMEN
Binding of sweet, umami, and bitter tastants to G protein-coupled receptors (GPCRs) in apical membranes of type II taste bud cells (TBCs) triggers action potentials that activate a voltage-gated nonselective ion channel to release ATP to gustatory nerves mediating taste perception. Although calcium homeostasis modulator 1 (CALHM1) is necessary for ATP release, the molecular identification of the channel complex that provides the conductive ATP-release mechanism suitable for action potential-dependent neurotransmission remains to be determined. Here we show that CALHM3 interacts with CALHM1 as a pore-forming subunit in a CALHM1/CALHM3 hexameric channel, endowing it with fast voltage-activated gating identical to that of the ATP-release channel in vivo. Calhm3 is co-expressed with Calhm1 exclusively in type II TBCs, and its genetic deletion abolishes taste-evoked ATP release from taste buds and GPCR-mediated taste perception. Thus, CALHM3, together with CALHM1, is essential to form the fast voltage-gated ATP-release channel in type II TBCs required for GPCR-mediated tastes.
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Canales de Calcio/fisiología , Activación del Canal Iónico/fisiología , Receptores Acoplados a Proteínas G/fisiología , Receptores Purinérgicos/fisiología , Percepción del Gusto/fisiología , Gusto/fisiología , Animales , Canales de Calcio/análisis , Femenino , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Transgénicos , Receptores Acoplados a Proteínas G/análisis , Receptores Purinérgicos/análisis , Transmisión Sináptica/fisiología , XenopusRESUMEN
Adenosine triphosphate (ATP) has been well established as an important extracellular ligand of autocrine signaling, intercellular communication, and neurotransmission with numerous physiological and pathophysiological roles. In addition to the classical exocytosis, non-vesicular mechanisms of cellular ATP release have been demonstrated in many cell types. Although large and negatively charged ATP molecules cannot diffuse across the lipid bilayer of the plasma membrane, conductive ATP release from the cytosol into the extracellular space is possible through ATP-permeable channels. Such channels must possess two minimum qualifications for ATP permeation: anion permeability and a large ion-conducting pore. Currently, five groups of channels are acknowledged as ATP-release channels: connexin hemichannels, pannexin 1, calcium homeostasis modulator 1 (CALHM1), volume-regulated anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR) anion channels), and maxi-anion channels (MACs). Recently, major breakthroughs have been made in the field by molecular identification of CALHM1 as the action potential-dependent ATP-release channel in taste bud cells, LRRC8s as components of VRACs, and SLCO2A1 as a core subunit of MACs. Here, the function and physiological roles of these five groups of ATP-release channels are summarized, along with a discussion on the future implications of understanding these channels.
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Adenosina Trifosfato/metabolismo , Conexinas/metabolismo , Animales , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Conexinas/genética , HumanosRESUMEN
Tolerance to bitter foods and its potentiation by repetitive exposure are commonly experienced and potentially underlie the consumption of bitter foods, but it remains unknown whether permissive and adaptive responses are general phenomena for bitter-tasting substances or specific to certain substances, and they have not been rigorously studied in mice. Here, we investigated the effects of prolonged exposure to a bitter compound on both recognition and rejection behaviors to the same compound in mice. Paired measurements of rejection (RjT) and apparent recognition (aRcT) thresholds were conducted using brief-access two-bottle choice tests before and after taste aversion conditioning, respectively. First, RjT was much higher than aRcT for the bitter amino acids L-tryptophan and L-isoleucine, which mice taste daily in their food, indicating strong acceptance of those familiar stimuli within the concentration range between RjT and aRcT. Next, we tested five other structurally dissimilar bitter compounds, to which mice were naive at the beginning of experiments: denatonium benzoate, quinine-HCl, caffeine, salicin, and epigallocatechin gallate. RjT was moderately higher than aRcT for all the compounds tested, indicating the presence of innate acceptance to these various, unfamiliar bitter stimuli in mice. Lastly, a 3-week forced exposure increased RjT for all the bitter compounds except salicin, demonstrating that mice acquire tolerance to a broad array of bitter compounds after long-term exposure to them. Although the underlying mechanisms remain to be determined, our studies provide behavioral evidence of innate and acquired tolerance to various bitter stimuli in mice, suggesting its generality among bitterants.
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Adaptación Fisiológica/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Isoleucina/farmacología , Percepción del Gusto/efectos de los fármacos , Triptófano/farmacología , Animales , Femenino , RatonesRESUMEN
Quercetin has multiple potential to control various cell function keeping our body condition healthy. In this review article, we describe the molecular mechanism on how quercetin exerts its action on blood pressure, neurite elongation and epithelial ion transport based from a viewpoint of cytosolic Cl- environments, which is recently recognized as an important signaling factor in various types of cells. Recent studies show various roles of cytosolic Cl- in regulation of blood pressure and neurite elongation, and prevention from bacterial and viral infection. We have found the stimulatory action of quercetin on Cl- transporter, Na+-K+-2Cl- cotransporter 1 (NKCC1; an isoform of NKCC), which has been recognized as one of the most interesting, fundamental actions of quercetin. In this review article, based on this stimulatory action of quercetin on NKCC1, we introduce the molecular mechanism of quercetin on: 1) blood pressure, 2) neurite elongation, and 3) epithelial Cl- secretion including tight junction forming in epithelial tissues. 1) Quercetin induces elevation of the cytosolic Cl- concentration via activation of NKCC1, leading to anti-hypertensive action by diminishing expression of epithelial Na+ channel (ENaC), a key ion channel involved in renal Na+ reabsorption, while quercetin has no effects on the blood pressure with normal salt intake. 2) Quercetin also has stimulatory effects on neurite elongation by elevating the cytosolic Cl- concentration via activation of NKCC1 due to tubulin polymerization facilitated through Cl--induced inhibition of GTPase. 3) Further, in lung airway epithelia quercetin stimulates Cl- secretion by increasing the driving force for Cl- secretion via elevation of the cytosolic Cl- concentration: this leads to water secretion, participating in prevention of our body from bacterial and viral infection. In addition to transcellular ion transport, quercetin regulates tight junction function via enhancement of tight junction integrity by modulating expression and assembling tight junction-forming proteins. Based on these observations, it is concluded that quercetin is a useful medicinal compound keeping our body to be in healthy condition.
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Antioxidantes/farmacología , Presión Sanguínea/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Neuritas/efectos de los fármacos , Quercetina/farmacología , Animales , Infecciones Bacterianas/prevención & control , Agua Corporal/metabolismo , Cloruros/metabolismo , Células Epiteliales/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Simportadores de Cloruro de Sodio-Potasio/genética , Simportadores de Cloruro de Sodio-Potasio/metabolismo , Uniones Estrechas/efectos de los fármacos , Virosis/prevención & controlRESUMEN
The increased production of reactive oxygen species (ROS) has been postulated to play a key role in the progression of nonalcoholic fatty liver disease (NAFLD). However, the source of ROS and mechanisms underlying the development of NAFLD have yet to be established. We observed a significant up-regulation of a minor isoform of NADPH oxidase, NOX1, in the liver of nonalcoholic steatohepatitis (NASH) patients as well as of mice fed a high-fat and high-cholesterol (HFC) diet for 8 weeks. In mice deficient in Nox1 (Nox1KO), increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 demonstrated in HFC diet-fed wild-type mice (WT) were significantly attenuated. Concomitantly, increased protein nitrotyrosine adducts, a marker of peroxynitrite-induced injury detected in hepatic sinusoids of WT, were significantly suppressed in Nox1KO. The expression of NOX1 mRNA was much higher in the fractions of enriched liver sinusoidal endothelial cells (LSECs) than in those of hepatocytes. In primary cultured LSECs, palmitic acid (PA) up-regulated the mRNA level of NOX1, but not of NOX2 or NOX4. The production of nitric oxide by LSECs was significantly attenuated by PA-treatment in WT but not in Nox1KO. When the in vitro relaxation of TWNT1, a cell line that originated from hepatic stellate cells, was assessed by the gel contraction assay, the relaxation of stellate cells induced by LSECs was attenuated by PA treatment. In contrast, the relaxation effect of LSECs was preserved in cells isolated from Nox1KO. Taken together, the up-regulation of NOX1 in LSECs may elicit peroxynitrite-mediated cellular injury and impaired hepatic microcirculation through the reduced bioavailability of nitric oxide. ROS derived from NOX1 may therefore constitute a critical component in the progression of NAFLD.
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Capilares/patología , Hígado/metabolismo , NADPH Oxidasa 1/metabolismo , NADPH Oxidasas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Alanina Transaminasa/sangre , Animales , Línea Celular , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Humanos , Hígado/irrigación sanguínea , Hígado/patología , Masculino , Ratones , Ratones Noqueados , NADPH Oxidasa 1/genética , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia ArribaRESUMEN
The detailed single-channel gating kinetics of mouse pannexin 1 (mPanx1) remains unknown, although mPanx1 is reported to be a voltage-activated anion-selective channel. We investigated characteristics of single-channel conductances and opening and closing rates of mPanx1 using patch-clamp techniques. The unitary current of mPanx1 shows outward rectification with single-channel conductances of ~20 pS for inward currents and ~80 pS for outward currents. The channel open time for outward currents (Cl- influx) increases linearly as the amplitude of single channel currents increases, while the open time for inward currents (Cl- efflux) is constant irrespective of changes in the current amplitude, as if the direction and amplitude of the unitary current regulates the open time. This is supported by further observations that replacement of extracellular Cl- with gluconate- diminishes the inward tail current (Cl- efflux) at a membrane potential of -100 mV due to the lowered outward current (gluconate- influx) at membrane potential of 100 mV. These results suggest that the direction and rate of charge-carrier movement regulate the open time of mPanx1, and that the previously reported voltage-dependence of Panx1 channel gating is not directly mediated by the membrane potential but rather by the direction and amplitude of currents through the channel.
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Conexinas/metabolismo , Activación del Canal Iónico , Proteínas del Tejido Nervioso/metabolismo , Animales , Línea Celular , Fenómenos Electrofisiológicos , Humanos , Cinética , RatonesRESUMEN
KEY POINTS: Calcium homeostasis modulator 1 (CALHM1), a new voltage-gated ATP- and Ca2+ -permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post-translationally regulated through the process of protein S-palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine-tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory. ABSTRACT: Emerging roles of CALHM1, a recently discovered voltage-gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post-translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S-palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance-voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent-resistant membranes. Our results comprehensively uncover a post-translational regulation of the voltage-dependent gating of CALHM1 by palmitoylation.
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Canales de Calcio/metabolismo , Activación del Canal Iónico , Microdominios de Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Canales de Calcio/genética , Células HeLa , Humanos , Lipoilación , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , XenopusRESUMEN
BACKGROUND/AIM: Epithelial Na+ channels (ENaC) play a crucial role in control of blood pressure by regulating renal Na+ reabsorption. Intracellular trafficking of ENaC is one of the key regulators of ENaC function, but a quantitative description of intracellular recycling of endogenously expressed ENaC is unavailable. We attempt here to provide a model for intracellular recycling after applying a protease inhibitor under hypotonic conditions. METHODS: We simulated the ENaC-mediated Na+ transport in renal epithelial A6 cells measured as short-circuit currents using a four-state mathematical ENaC trafficking model. RESULTS: We developed a four-state mathematical model of ENaC trafficking in the cytosol of renal epithelial cells that consists of: an insertion state of ENaC that can be trafficked to the apical membrane state (insertion rate); an apical membrane state of ENaC conducting Na+ across the apical membrane; a recycling state containing ENaC that are retrieved from the apical membrane state (endocytotic rate) and then to the insertion state (recycling rate) communicating with the apical membrane state or to a degradation state (degradation rate). We studied the effect of aprotinin (a protease inhibitor) blocking protease-induced cleavage of the extracellular loop of γ ENaC subunit on the rates of intracellular ENaC trafficking using the above-defined four-state mathematical model of ENaC trafficking and the recycling number relative to ENaC staying in the apical membrane. We found that aprotinin significantly reduced the insertion rate of ENaC to the apical membrane by 40%, the recycling rate of ENaC by 81%, the cumulative time of an individual ENaC staying in the apical membrane by 32%, the cumulative life-time after the first endocytosis of ENaC by 25%, and the cumulative Na+ absorption by 31%. The most interesting result of the present study is that cleavage of ENaC affects the intracellular ENaC trafficking rate and determines the residency time of ENaC, indicating that more active cleaved ENaCs stay longer at the apical membrane contributing to transcellular Na+ transport via an increase in recycling of ENaC to the apical membrane. CONCLUSION: The extracellular protease-induced cleavage of the extracellular loop of γ ENaC subunit increases transcellular epithelial Na+ transport by elevating the recycling rate of ENaC due to an increase in the recycling rate of ENaCs associated with increases in the insertion rate of ENaC.