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1.
J Neurosci ; 40(42): 7999-8024, 2020 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-32928889

RESUMEN

In multipolar vertebrate neurons, action potentials (APs) initiate close to the soma, at the axonal initial segment. Invertebrate neurons are typically unipolar with dendrites integrating directly into the axon. Where APs are initiated in the axons of invertebrate neurons is unclear. Voltage-gated sodium (NaV) channels are a functional hallmark of the axonal initial segment in vertebrates. We used an intronic Minos-Mediated Integration Cassette to determine the endogenous gene expression and subcellular localization of the sole NaV channel in both male and female Drosophila, para Despite being the only NaV channel in the fly, we show that only 23 ± 1% of neurons in the embryonic and larval CNS express para, while in the adult CNS para is broadly expressed. We generated a single-cell transcriptomic atlas of the whole third instar larval brain to identify para expressing neurons and show that it positively correlates with markers of differentiated, actively firing neurons. Therefore, only 23 ± 1% of larval neurons may be capable of firing NaV-dependent APs. We then show that Para is enriched in an axonal segment, distal to the site of dendritic integration into the axon, which we named the distal axonal segment (DAS). The DAS is present in multiple neuron classes in both the third instar larval and adult CNS. Whole cell patch clamp electrophysiological recordings of adult CNS fly neurons are consistent with the interpretation that Nav-dependent APs originate in the DAS. Identification of the distal NaV localization in fly neurons will enable more accurate interpretation of electrophysiological recordings in invertebrates.SIGNIFICANCE STATEMENT The site of action potential (AP) initiation in invertebrates is unknown. We tagged the sole voltage-gated sodium (NaV) channel in the fly, para, and identified that Para is enriched at a distal axonal segment. The distal axonal segment is located distal to where dendrites impinge on axons and is the likely site of AP initiation. Understanding where APs are initiated improves our ability to model neuronal activity and our interpretation of electrophysiological data. Additionally, para is only expressed in 23 ± 1% of third instar larval neurons but is broadly expressed in adults. Single-cell RNA sequencing of the third instar larval brain shows that para expression correlates with the expression of active, differentiated neuronal markers. Therefore, only 23 ± 1% of third instar larval neurons may be able to actively fire NaV-dependent APs.


Asunto(s)
Segmento Inicial del Axón/metabolismo , Proteínas de Drosophila/biosíntesis , Drosophila/metabolismo , Neuronas/metabolismo , Canales de Sodio/biosíntesis , Canales de Sodio Activados por Voltaje/biosíntesis , Potenciales de Acción/fisiología , Animales , Axones/fisiología , Dendritas/metabolismo , Proteínas de Drosophila/genética , Fenómenos Electrofisiológicos , Electrorretinografía , Expresión Génica/genética , Larva , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Técnicas de Placa-Clamp , Canales de Sodio/genética , Transcriptoma , Canales de Sodio Activados por Voltaje/genética
2.
J Neurochem ; 147(4): 454-476, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30182387

RESUMEN

As the peripheral sympathoadrenal axis is tightly controlled by the cortex via hypothalamus and brain stem, the central pathological features of Hunting's disease, (HD) that is, deposition of mutated huntingtin and synaptic dysfunctions, could also be expressed in adrenal chromaffin cells. To test this hypothesis we here present a thorough investigation on the pathological and functional changes undergone by chromaffin cells (CCs) from 2-month (2 m) to 7-month (7 m) aged wild-type (WT) and R6/1 mouse model of Huntington's disease (HD), stimulated with acetylcholine (ACh) or high [K+ ] (K+ ). In order to do this, we used different techniques such as inmunohistochemistry, patch-clamp, and amperometric recording. With respect to WT cells, some of the changes next summarized were already observed in HD mice at a pre-disease stage (2 m); however, they were more pronounced at 7 m when motor deficits were clearly established, as follows: (i) huntingtin over-expression as nuclear aggregates in CCs; (ii) smaller CC size with decreased dopamine ß-hydroxylase expression, indicating lesser number of chromaffin secretory vesicles; (iii) reduced adrenal tissue catecholamine content; (iv) reduced Na+ currents with (v) membrane hyperpolarization and reduced ACh-evoked action potentials; (v) reduced [Ca2+ ]c transients with faster Ca2+ clearance; (vi) diminished quantal secretion with smaller vesicle quantal size; (vii) faster kinetics of the exocytotic fusion pore, pore expansion, and closure. On the basis of these data, the hypothesis is here raised in the sense that nuclear deposition of mutated huntingtin in adrenal CCs of R6/1 mice could be primarily responsible for poorer Na+ channel expression and function, giving rise to profound depression of cell excitability, altered Ca2+ handling and exocytosis. OPEN PRACTICES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14201.


Asunto(s)
Células Cromafines/metabolismo , Células Cromafines/patología , Exocitosis , Proteína Huntingtina/biosíntesis , Proteína Huntingtina/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Médula Suprarrenal/metabolismo , Médula Suprarrenal/patología , Animales , Catecolaminas/metabolismo , Humanos , Enfermedad de Huntington/psicología , Cinética , Masculino , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Trastornos del Movimiento/etiología , Trastornos del Movimiento/fisiopatología , Mutación/genética , Desempeño Psicomotor , Canales de Sodio/biosíntesis , Vesículas Sinápticas/patología
3.
Anesthesiology ; 128(6): 1151-1166, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29509584

RESUMEN

BACKGROUND: Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils. METHODS: Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp. RESULTS: Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2. CONCLUSIONS: Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.


Asunto(s)
Adhesión Celular/fisiología , Quimiotaxis/fisiología , Riñón/metabolismo , Isquemia Miocárdica/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/biosíntesis , Neutrófilos/metabolismo , Canales de Sodio/biosíntesis , Migración Transendotelial y Transepitelial/fisiología , Animales , Adhesión Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Expresión Génica , Humanos , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Lidocaína/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Isquemia Miocárdica/tratamiento farmacológico , Canal de Sodio Activado por Voltaje NAV1.3/genética , Neutrófilos/efectos de los fármacos , Canales de Sodio/genética , Migración Transendotelial y Transepitelial/efectos de los fármacos
4.
Neuropharmacology ; 113(Pt A): 480-489, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27816501

RESUMEN

Abnormal expressions of sodium channel SCN1A and SCN3A genes alter neural excitability that are believed to contribute to the pathogenesis of epilepsy, a long-term risk of recurrent seizures. Ketogenic diet (KD), a high-fat and low-carbohydrate treatment for difficult-to-control (refractory) epilepsy in children, has been suggested to reverse gene expression patterns. Here, we reveal a novel role of GAPDH on the posttranscriptional regulation of mouse Scn1a and Scn3a expressions under seizure and KD conditions. We show that GAPDH binds to a conserved region in the 3' UTRs of human and mouse SCN1A and SCN3A genes, which decreases and increases genes' expressions by affecting mRNA stability through SCN1A 3' UTR and SCN3A 3' UTR, respectively. In seizure mice, the upregulation and phosphorylation of GAPDH enhance its binding to the 3' UTR, which lead to downregulation of Scn1a and upregulation of Scn3a. Furthermore, administration of KD generates ß-hydroxybutyric acid which rescues the abnormal expressions of Scn1a and Scn3a by weakening the GAPDH's binding to the element. Taken together, these data suggest that GAPDH-mediated expression regulation of sodium channel genes may be associated with epilepsy and the anticonvulsant action of KD.


Asunto(s)
Dieta Cetogénica , Gliceraldehído-3-Fosfato Deshidrogenasas/fisiología , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.3/genética , Convulsiones/dietoterapia , Convulsiones/genética , Canales de Sodio/genética , Animales , Línea Celular Tumoral , Dieta Cetogénica/métodos , Células HEK293 , Humanos , Masculino , Ratones , Canal de Sodio Activado por Voltaje NAV1.1/biosíntesis , Canal de Sodio Activado por Voltaje NAV1.3/biosíntesis , Unión Proteica/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Convulsiones/metabolismo , Canales de Sodio/biosíntesis
5.
Heart Rhythm ; 13(4): 983-91, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26711798

RESUMEN

BACKGROUND: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly patients and has been correlated with enhanced age-dependent atrial fibrosis. Reversal of atrial fibrosis has been proposed as therapeutic strategy to suppress AF. OBJECTIVE: To test the ability of relaxin to reverse age-dependent atrial fibrosis and suppress AF. METHODS: Aged F-344 rats (24 months old) were treated with subcutaneous infusion of vehicle or relaxin (0.4 mg/kg/day) for 2 weeks. Rat hearts were excised, perfused on a Langendorff apparatus, and stained with voltage and Ca(2+) indicator dyes. Optical mapping and programmed electrical stimulation was used to test arrhythmia vulnerability and changes in electrophysiological characteristics. Changes in protein expression and Na(+) current density (INa) were measured by tissue immunofluorescence and whole-cell patch clamp technique. RESULTS: In aged rats, sustained AF was readily induced with a premature pulse (n = 7/8) and relaxin treatment suppressed sustained AF by a premature impulse or burst pacing (n = 1/6) (P < .01). Relaxin significantly increased atrial action potential conduction velocity and decreased atrial fibrosis. Relaxin treatment increased Nav1.5 expression (n = 6; 36% ± 10%) and decreased total collagen and collagen I (n = 5-6; 55%-66% ± 15%) in aged atria (P < .05) and decreased collagen I and III and TGF-ß1 mRNA (P < .05). Voltage-clamp experiments demonstrated that relaxin treatment (100 nM for 2 days) increased atrial INa by 46% ± 4% (n = 12-13/group, P < .02). CONCLUSION: Relaxin suppresses AF through an increase in atrial conduction velocity by decreasing atrial fibrosis and increasing INa. These data provide compelling evidence that relaxin may serve as an effective therapy to manage AF in geriatric patients by reversing fibrosis and modulating cardiac ionic currents.


Asunto(s)
Fibrilación Atrial/tratamiento farmacológico , Atrios Cardíacos/metabolismo , Sistema de Conducción Cardíaco/fisiopatología , Miocitos Cardíacos/metabolismo , Relaxina/farmacología , Canales de Sodio/biosíntesis , Regulación hacia Arriba , Potenciales de Acción/efectos de los fármacos , Animales , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Fibrosis/patología , Fibrosis/fisiopatología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/patología , Sistema de Conducción Cardíaco/efectos de los fármacos , Masculino , Miocitos Cardíacos/patología , Ratas , Ratas Endogámicas F344 , Ratas Endogámicas WKY , Canales de Sodio/efectos de los fármacos
6.
Mol Hum Reprod ; 21(10): 816-24, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26134120

RESUMEN

Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 µM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability.


Asunto(s)
Miometrio/metabolismo , Tercer Trimestre del Embarazo/metabolismo , Canales de Sodio/fisiología , Sodio/metabolismo , Contracción Uterina/fisiología , Adulto , Biopsia , Femenino , Gadolinio/farmacología , Perfilación de la Expresión Génica , Humanos , Canales Iónicos , Transporte Iónico/fisiología , Potenciales de la Membrana/fisiología , Proteínas de la Membrana , Miometrio/citología , Técnicas de Placa-Clamp , Embarazo , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/biosíntesis , Canales de Sodio/genética
7.
PLoS One ; 9(6): e98808, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24945283

RESUMEN

The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE) and calcium (EEEE) selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM) was lowered to 0.1 mM and were inhibited (>40% to >90%) with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (<30%) to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 µM mibefradil or 100 µM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.


Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Canales de Sodio/biosíntesis , Sodio/metabolismo , Canales de Calcio/biosíntesis , Canales de Calcio/metabolismo , Membrana Celular/química , Células HEK293 , Humanos , Canales Iónicos , Proteínas de la Membrana , Nifedipino/farmacología , Técnicas de Placa-Clamp , Canales de Sodio/metabolismo , Verapamilo/administración & dosificación
8.
PLoS Genet ; 10(3): e1004238, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24675786

RESUMEN

As in many species, gustatory pheromones regulate the mating behavior of Drosophila. Recently, several ppk genes, encoding ion channel subunits of the DEG/ENaC family, have been implicated in this process, leading to the identification of gustatory neurons that detect specific pheromones. In a subset of taste hairs on the legs of Drosophila, there are two ppk23-expressing, pheromone-sensing neurons with complementary response profiles; one neuron detects female pheromones that stimulate male courtship, the other detects male pheromones that inhibit male-male courtship. In contrast to ppk23, ppk25, is only expressed in a single gustatory neuron per taste hair, and males with impaired ppk25 function court females at reduced rates but do not display abnormal courtship of other males. These findings raised the possibility that ppk25 expression defines a subset of pheromone-sensing neurons. Here we show that ppk25 is expressed and functions in neurons that detect female-specific pheromones and mediates their stimulatory effect on male courtship. Furthermore, the role of ppk25 and ppk25-expressing neurons is not restricted to responses to female-specific pheromones. ppk25 is also required in the same subset of neurons for stimulation of male courtship by young males, males of the Tai2 strain, and by synthetic 7-pentacosene (7-P), a hydrocarbon normally found at low levels in both males and females. Finally, we unexpectedly find that, in females, ppk25 and ppk25-expressing cells regulate receptivity to mating. In the absence of the third antennal segment, which has both olfactory and auditory functions, mutations in ppk25 or silencing of ppk25-expressing neurons block female receptivity to males. Together these results indicate that ppk25 identifies a functionally specialized subset of pheromone-sensing neurons. While ppk25 neurons are required for the responses to multiple pheromones, in both males and females these neurons are specifically involved in stimulating courtship and mating.


Asunto(s)
Proteínas de Drosophila/biosíntesis , Neuronas/metabolismo , Feromonas/genética , Conducta Sexual Animal , Canales de Sodio/biosíntesis , Animales , Cortejo , Drosophila , Proteínas de Drosophila/genética , Femenino , Regulación de la Expresión Génica , Masculino , Mutación , Canales de Sodio/genética
9.
Circulation ; 126(9): 1058-66, 2012 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-22837163

RESUMEN

BACKGROUND: Notch signaling has previously been shown to play an essential role in regulating cell fate decisions and differentiation during cardiogenesis in many systems including Drosophila, Xenopus, and mammals. We hypothesized that Notch may also be involved in directing the progressive lineage restriction of cardiomyocytes into specialized conduction cells. METHODS AND RESULTS: In hearts where Notch signaling is activated within the myocardium from early development onward, Notch promotes a conduction-like phenotype based on ectopic expression of conduction system-specific genes and cell autonomous changes in electrophysiology. With the use of an in vitro assay to activate Notch in newborn cardiomyocytes, we observed global changes in the transcriptome, and in action potential characteristics, consistent with reprogramming to a conduction-like phenotype. CONCLUSIONS: Notch can instruct the differentiation of chamber cardiac progenitors into specialized conduction-like cells. Plasticity remains in late-stage cardiomyocytes, which has potential implications for engineering of specialized cardiovascular tissues.


Asunto(s)
Nodo Atrioventricular/citología , Regulación del Desarrollo de la Expresión Génica , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Receptor Notch1/fisiología , Potenciales de Acción , Adenoviridae/genética , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula , Contactina 2/biosíntesis , Contactina 2/genética , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Ratones , Miocitos Cardíacos/ultraestructura , Canal de Sodio Activado por Voltaje NAV1.5 , Plasticidad Neuronal , Técnicas de Placa-Clamp , Fenotipo , Ramos Subendocárdicos/citología , Receptor Notch1/genética , Proteínas Recombinantes de Fusión/fisiología , Transducción de Señal/fisiología , Canales de Sodio/biosíntesis , Canales de Sodio/genética , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Factor de Transcripción HES-1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética
10.
Clin Exp Allergy ; 42(7): 1028-39, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22702502

RESUMEN

BACKGROUND: Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. OBJECTIVES: The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. METHODS: Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. RESULTS: Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway. CONCLUSIONS: This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.


Asunto(s)
Peroxidasa del Eosinófilo/metabolismo , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Mucosa Nasal/metabolismo , Rinitis Alérgica Estacional/metabolismo , Canales de Sodio/biosíntesis , Canales Iónicos Sensibles al Ácido , Adolescente , Adulto , Biopsia , Células Cultivadas , Células Epiteliales/patología , Femenino , Humanos , Ácido Láctico/farmacología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mucosa Nasal/patología , Reacción en Cadena de la Polimerasa , Rinitis Alérgica Estacional/patología
11.
Neurosci Lett ; 516(2): 197-201, 2012 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-22708125

RESUMEN

Acid-sensing ion channel 2 (ASIC2) is a member of the degenerin/epithelial sodium channel superfamily, presumably involved mechanosensation. Expression of ASIC2 has been detected in mechanosensory neurons as well as in both axons and Schwann-like cells of cutaneous mechanoreceptors. In these studies we analysed expression of ASIC2 in the cutaneous sensory corpuscles of Macaca fascicularis using immunohistochemistry and laser confocal-scanner microscopy. ASIC2 immunoreactivity was detected in both Meissner and Pacinian corpuscles. It was found to co-localize with neuron-specific enolase and RT-97, but not with S100 protein, demonstrating that ASIC2 expression is restricted to axons supplying mechanoreceptors. These results demonstrate for the first time the presence of the protein ASIC2 in cutaneous rapidly adapting low-threshold mechanoreceptors of monkey, suggesting a role of this ion channel in touch sense.


Asunto(s)
Mecanorreceptores/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Corpúsculos de Pacini/metabolismo , Canales de Sodio/biosíntesis , Percepción del Tacto/fisiología , Canales Iónicos Sensibles al Ácido , Animales , Axones/metabolismo , Inmunohistoquímica , Macaca fascicularis , Masculino , Microscopía Confocal , Proteínas del Tejido Nervioso/análisis , Canales de Sodio/análisis
12.
Brain Res ; 1463: 30-41, 2012 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-22608073

RESUMEN

Sub- and near-threshold voltage-dependent Na+ currents (VDSCs) are of major importance in determining the electrical properties of medial entorhinal cortex (mEC) layer-II neurons. Developmental changes in the ability of mEC layer-II stellate cells (SCs) to generate Na+ -dependent, subthreshold electrical events have been reported between P14 and P18. In this study we examined the modifications occurring in the various components of VDSCs during postnatal development of mEC SCs. The transient, resurgent, and persistent Na+ currents (I(NaT), I(NaR), and I(NaP), respectively) showed distinct patterns of developmental expression in the time window considered (P5 to P24-27). All three currents prominently and steeply increased in absolute amplitude and conductance from P5 to at least P16. However, capacitive charge accumulation, an index of membrane surface area, also markedly increased in the same time window, and in the case of I(NaT) the specific conductance per unit of accumulated capacitive charge remained relatively constant. By contrast, specific I(NaR) and I(NaP) conductances showed a significant tendency to increase, especially from P5 to P18. Neither I(NaR) nor I(NaP) represented a constant fraction of the total Na+ current at all developmental ages. Indeed, detectable levels of I(NaR) and I(NaP) were present in only ~20% and ~70%, respectively, of the cells on P5, and were observed in all cells only from P10 onwards. Moreover, the average I(NaR)-to-I(NaT) conductance ratio increased steadily from ~0.004 (P5) up to a plateau level of ~0.05 (P22+), whereas the I(NaP)-to-I(NaT) conductance ratio increased only from ~0.009 on P5 to ~0.02 on P22+. The relative increase in conductance ratio from P5 to P22 was significantly greater for I(NaR) than for I(NaP), indicating that I(NaR) expression starts later than that of I(NaP). These findings show that in mEC layer-II SCs the single functional components of the VDSC are regulated differentially from each other as far as their developmental expression is concerned.


Asunto(s)
Corteza Entorrinal/crecimiento & desarrollo , Corteza Entorrinal/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas/fisiología , Canales de Sodio/biosíntesis , Canales de Sodio/genética , Potenciales de Acción/genética , Animales , Animales Recién Nacidos , Corteza Entorrinal/citología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Tiempo de Reacción/genética , Canales de Sodio/fisiología
13.
Int J Biol Sci ; 8(5): 630-9, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22553463

RESUMEN

Aberrant protein glycosylation plays major roles in neurodegenerative diseases, including Parkinson's disease (PD). Glycoproteomics showed that the glycosylation of sodium channel ß4 was significantly increased in human brain tissue. ß4-specific antibodies reacted in immunoblot assays with the 35- and 38-kDa bands from the membrane fractions isolated from neonatal PD transgenic mice but only with the 35-kDa band of the neonatal wild-type mice. The size of the 38-kDa immunoreactive protein is in close agreement with previously reported, suggesting heavy glycosylation of this protein in adult wild-type and neonatal PD transgenic brain tissues. However, the neonatal wild-type mice membrane fractions only contained the 35-kDa immunoreactive protein, and the additional 38-kDa band was not shown until postnatal day 7. Enzymatic deglycosylation of the membrane preparations only converted the 38-kDa band into a faster migrating protein, which was consistent with heavy glycosylation of this protein. The glycosylated state of ß4 was developmentally regulated and was altered in disease state. Neurite outgrowth assay demonstrated that overexpression of deglycosylated mutant ß4-MUT accelerated neurite extension and increased the number of filopodia-like protrusions, when compared with ß4-WT and the vector. These results suggest that extensive glycosylation of ß4 subunit play roles in morphological changes, and the altered glycosylation may be involved in the pathogenesis of PD.


Asunto(s)
Neuritas/fisiología , Canales de Sodio/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Glicosilación , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Neuritas/patología , Enfermedad de Parkinson/fisiopatología , Seudópodos/fisiología , Canales de Sodio/biosíntesis , Subunidad beta-4 de Canal de Sodio Activado por Voltaje
14.
Proc Natl Acad Sci U S A ; 109(31): E2134-43, 2012 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-22509027

RESUMEN

The cardiac electrical impulse depends on an orchestrated interplay of transmembrane ionic currents in myocardial cells. Two critical ionic current mechanisms are the inwardly rectifying potassium current (I(K1)), which is important for maintenance of the cell resting membrane potential, and the sodium current (I(Na)), which provides a rapid depolarizing current during the upstroke of the action potential. By controlling the resting membrane potential, I(K1) modifies sodium channel availability and therefore, cell excitability, action potential duration, and velocity of impulse propagation. Additionally, I(K1)-I(Na) interactions are key determinants of electrical rotor frequency responsible for abnormal, often lethal, cardiac reentrant activity. Here, we have used a multidisciplinary approach based on molecular and biochemical techniques, acute gene transfer or silencing, and electrophysiology to show that I(K1)-I(Na) interactions involve a reciprocal modulation of expression of their respective channel proteins (Kir2.1 and Na(V)1.5) within a macromolecular complex. Thus, an increase in functional expression of one channel reciprocally modulates the other to enhance cardiac excitability. The modulation is model-independent; it is demonstrable in myocytes isolated from mouse and rat hearts and with transgenic and adenoviral-mediated overexpression/silencing. We also show that the post synaptic density, discs large, and zonula occludens-1 (PDZ) domain protein SAP97 is a component of this macromolecular complex. We show that the interplay between Na(v)1.5 and Kir2.1 has electrophysiological consequences on the myocardium and that SAP97 may affect the integrity of this complex or the nature of Na(v)1.5-Kir2.1 interactions. The reciprocal modulation between Na(v)1.5 and Kir2.1 and the respective ionic currents should be important in the ability of the heart to undergo self-sustaining cardiac rhythm disturbances.


Asunto(s)
Potenciales de Acción , Arritmias Cardíacas/mortalidad , Regulación de la Expresión Génica , Potenciales de la Membrana , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/metabolismo , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Sodio/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Homólogo 1 de la Proteína Discs Large , Silenciador del Gen , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.5 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Canales de Potasio de Rectificación Interna/genética , Ratas , Ratas Sprague-Dawley , Ratas Transgénicas , Canales de Sodio/genética , Proteína de la Zonula Occludens-1
15.
J Biol Chem ; 287(18): 14443-55, 2012 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-22399291

RESUMEN

Acid-sensing ion channels (ASICs) are non-selective cation channels activated by extracellular acidosis associated with many physiological and pathological conditions. A detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In this study, we examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp(107)-Arg(153)) and human ASIC1a (Asp(107)-Arg(160)) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt bridge is essential for functional expression of ASICs in a pH sensing-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis, and further mutagenesis studies suggested a high stability and structural constrain of the salt bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Activación del Canal Iónico/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Canales de Sodio/biosíntesis , Canales Iónicos Sensibles al Ácido , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Secundaria de Proteína , Ratas , Canales de Sodio/química , Canales de Sodio/genética
16.
J Biol Chem ; 287(18): 15044-53, 2012 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-22408255

RESUMEN

The small-diameter (<25 µm) and large-diameter (>30 µm) sensory neurons of the dorsal root ganglion (DRG) express distinct combinations of tetrodotoxin sensitive and tetrodotoxin-resistant Na(+) channels that underlie the unique electrical properties of these neurons. In vivo, these Na(+) channels are formed as complexes of pore-forming α and auxiliary ß subunits. The goal of this study was to investigate the expression of ß subunits in DRG sensory neurons. Quantitative single-cell RT-PCR revealed that ß subunit mRNA is differentially expressed in small (ß(2) and ß(3)) and large (ß(1) and ß(2)) DRG neurons. This raises the possibility that ß subunit availability and Na(+) channel composition and functional regulation may differ in these subpopulations of sensory neurons. To further explore these possibilities, we quantitatively compared the mRNA expression of the ß subunit with that of Na(v)1.7, a TTX-sensitive Na(+) channel widely expressed in both small and large DRG neurons. Na(v)1.7 and ß subunit mRNAs were significantly correlated in small (ß(2) and ß(3)) and large (ß(1) and ß(2)) DRG neurons, indicating that these subunits are coexpressed in the same populations. Co-immunoprecipitation and immunocytochemistry indicated that Na(v)1.7 formed stable complexes with the ß(1)-ß(3) subunits in vivo and that Na(v)1.7 and ß(3) co-localized within the plasma membranes of small DRG neurons. Heterologous expression studies showed that ß(3) induced a hyperpolarizing shift in Na(v)1.7 activation, whereas ß(1) produced a depolarizing shift in inactivation and faster recovery. The data indicate that ß(3) and ß(1) subunits are preferentially expressed in small and large DRG neurons, respectively, and that these auxiliary subunits differentially regulate the gating properties of Na(v)1.7 channels.


Asunto(s)
Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/fisiología , Activación del Canal Iónico/fisiología , Proteínas del Tejido Nervioso/biosíntesis , Células Receptoras Sensoriales/metabolismo , Canales de Sodio/biosíntesis , Animales , Membrana Celular/metabolismo , Ganglios Espinales/citología , Inmunohistoquímica/métodos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Receptoras Sensoriales/citología
17.
J Neurosci ; 32(13): 4665-74, 2012 Mar 28.
Artículo en Inglés | MEDLINE | ID: mdl-22457513

RESUMEN

Detection of specific female pheromones stimulates courtship behavior in Drosophila melanogaster males, but the chemosensory molecules, cells, and mechanisms involved remain poorly understood. Here we show that ppk25, a DEG/ENaC ion channel subunit required for normal male response to females, is expressed at highest levels in a single sexually dimorphic gustatory neuron of most taste hairs on legs and wings, but not in neurons that detect courtship-inhibiting pheromones or food. Synaptic inactivation of ppk25-expressing neurons, or knockdown of ppk25 expression in all gustatory neurons, significantly impairs male response to females, whereas gustatory expression of ppk25 rescues the courtship behavior of ppk25 mutant males. Remarkably, the only other detectable albeit significantly weaker expression of ppk25 occurs in olfactory neurons implicated in modulation of courtship behavior. However, expression of ppk25 in olfactory neurons is not required for male courtship under our experimental conditions. These data show that ppk25 functions specifically in peripheral taste neurons involved in activation of courtship behavior, an unexpected function for this type of channel. Furthermore, our work identifies a small subset of gustatory neurons with an essential role in activation of male courtship behavior, most likely in response to female pheromones.


Asunto(s)
Células Quimiorreceptoras/fisiología , Cortejo , Proteínas de Drosophila/fisiología , Canales de Sodio/fisiología , Animales , Células Quimiorreceptoras/metabolismo , Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Técnicas de Silenciamiento del Gen/psicología , Masculino , Imagen Molecular/métodos , Feromonas/fisiología , Canales de Sodio/biosíntesis , Canales de Sodio/genética
18.
Ann Neurol ; 71(2): 186-94, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22367990

RESUMEN

OBJECTIVE: Cerebellar dysfunction in multiple sclerosis (MS) contributes significantly to disability, is relatively refractory to symptomatic therapy, and often progresses despite treatment with disease-modifying agents. We previously observed that sodium channel Nav1.8, whose expression is normally restricted to the peripheral nervous system, is present in cerebellar Purkinje neurons in a mouse model of MS (experimental autoimmune encephalomyelitis [EAE]) and in humans with MS. Here, we tested the hypothesis that upregulation of Nav1.8 in cerebellum in MS and EAE has functional consequences contributing to symptom burden. METHODS: Electrophysiology and behavioral assessment were performed in a new transgenic mouse model overexpressing Nav1.8 in Purkinje neurons. We also measured EAE symptom progression in mice lacking Nav1.8 compared to wild-type littermates. Finally, we administered the Nav1.8-selective blocker A803467 in the context of previously established EAE to determine reversibility of MS-like deficits. RESULTS: We report that, in the context of an otherwise healthy nervous system, ectopic expression of Nav1.8 in Purkinje neurons alters their electrophysiological properties, and disrupts coordinated motor behaviors. Additionally, we show that Nav1.8 expression contributes to symptom development in EAE. Finally, we demonstrate that abnormal patterns of Purkinje neuron firing and MS-like deficits in EAE can be partially reversed by pharmacotherapy using a Nav1.8-selective blocker. INTERPRETATION: Our results add to the evidence that a channelopathy contributes to cerebellar dysfunction in MS. Our data suggest that Nav1.8-specific blockers, when available for humans, merit study in MS.


Asunto(s)
Enfermedades Cerebelosas/fisiopatología , Canalopatías/fisiopatología , Encefalomielitis Autoinmune Experimental/fisiopatología , Esclerosis Múltiple/fisiopatología , Compuestos de Anilina/uso terapéutico , Animales , Enfermedades Cerebelosas/genética , Cerebelo/citología , Cerebelo/metabolismo , Cerebelo/patología , Canalopatías/genética , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Furanos/uso terapéutico , Ratones , Ratones Transgénicos , Esclerosis Múltiple/genética , Canal de Sodio Activado por Voltaje NAV1.8 , Células de Purkinje/patología , Células de Purkinje/fisiología , Bloqueadores de los Canales de Sodio/uso terapéutico , Canales de Sodio/biosíntesis , Canales de Sodio/genética , Canales de Sodio/metabolismo , Regulación hacia Arriba/genética
19.
Neuroscience ; 206: 237-44, 2012 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-22260870

RESUMEN

We reported previously that sensory neurons isolated from mice with a heterozygous mutation of the Nf1 gene (Nf1+/-) exhibited greater excitability and increased sodium current densities compared with wildtype mice. This raises the question as to whether the increased current density resulted from post-translational modifications or increased expression of sodium channels. Quantitative real-time polymerase chain reaction was used to measure expression levels of the nine different voltage-gated sodium channel α subunits and the four associated auxiliary ß subunits in the dorsal root ganglia (DRG) obtained from wildtype and Nf1+/- mice. The Relative Expression Software Tool indicated that Nav1.1, Nav1.3, Nav1.7, and Nav1.8 were significantly elevated in DRG isolated from Nf1+/- mice. Expression of Nav1.2, Nav1.5, Nav1.6, and Nav1.9 were not significantly altered. The gene transcript for Nav1.4 was not detected. There were no significant changes in the relative expression levels of ß subunits. The Nav1.9 subtype was the most abundant with Nav1.7 and Nav1.8 being the next most abundant subtypes, whereas Nav1.3 was relatively less abundant. For the ß subunits, ß1 was by far the most abundant subtype. These results demonstrate that the increased expression levels of Nav1.7, Nav1.8, and perhaps Nav1.1 in the Nf1+/- DRG make the largest contribution to the increased sodium current density and thus give rise to the enhanced excitability. Though the mechanisms by which many people with NF1 experience increased pain have not been elucidated, these abnormal painful states may involve elevated expression of specific sodium channel subtypes in small diameter nociceptive sensory neurons.


Asunto(s)
Ganglios Espinales/metabolismo , Genes de Neurofibromatosis 1 , ARN Mensajero/biosíntesis , Canales de Sodio/biosíntesis , Canales de Sodio/genética , Animales , Ratones , Ratones Endogámicos C57BL , Mutación , Canal de Sodio Activado por Voltaje NAV1.1 , Canal de Sodio Activado por Voltaje NAV1.3 , Canal de Sodio Activado por Voltaje NAV1.7 , Canal de Sodio Activado por Voltaje NAV1.8 , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
20.
Heart Rhythm ; 9(4): 600-7, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22100711

RESUMEN

BACKGROUND: Reduced expression of connexin43 (Cx43) and sodium channel (Nav1.5) and increased expression of collagen (fibrosis) are important determinants of impulse conduction in the heart. OBJECTIVE: To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx43 knockout mice with and without inducible ventricular tachycardia (VT) were compared through electrophysiology and immunohistochemistry. METHODS: Cx43(CreER(T)/fl) mice were induced with tamoxifen and killed after 2 weeks. Epicardial activation mapping was performed on Langendorff-perfused hearts, and arrhythmia vulnerability was tested. Mice were divided into arrhythmogenic (VT+; n = 13) and nonarrhythmogenic (VT-; n = 10) animals, and heart tissue was analyzed for Cx43, Nav1.5, and fibrosis. RESULTS: VT+ mice had decreased Cx43 expression with increased global, but not local, heterogeneity of Cx43 than did VT- mice. Nav1.5-immunoreactive protein expression was lower in VT+ than in VT- mice, specifically at sites devoid of Cx43. Levels of fibrosis were similar between VT- and VT+ mice. QRS duration was increased and epicardial activation was more dispersed in VT+ mice than in VT- mice. The effective refractory period was similar between the 2 groups. Premature stimulation resulted in a more severe conduction slowing in VT+ than in VT- hearts in the right ventricle. Separate patch-clamp experiments in isolated rat ventricular myocytes confirmed that the loss of Cx43 expression correlated with the decreased sodium current amplitude. CONCLUSIONS: Global heterogeneity in Cx43 expression and concomitant heterogeneous downregulation of sodium-channel protein expression and sodium current leads to slowed and dispersed conduction, which sensitizes the heart for ventricular arrhythmias.


Asunto(s)
Arritmias Cardíacas/genética , Colágeno/biosíntesis , Conexina 43/biosíntesis , Fibrosis/genética , Canales de Sodio/biosíntesis , Taquicardia Ventricular/genética , Animales , Arritmias Cardíacas/patología , Colágeno/genética , Conexina 43/genética , Modelos Animales de Enfermedad , Femenino , Fibrosis/patología , Masculino , Ratones , Ratones Noqueados , Células Musculares , Canal de Sodio Activado por Voltaje NAV1.5 , Riesgo , Canales de Sodio/genética , Taquicardia Ventricular/patología
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