RESUMEN
INTRODUCTION: ICl,stretch have been reported to be involved in the development of atrial fibrillation, so we observed the changes of transcription and translation levels of ICl,stretch in isolated atrial myocardium of heart failure canine models. MATERIAL AND METHODS: In the control group (N = 10), five dogs were untreated and the other five received sham operation, while dogs in the heart failure group (N = 10) were implanted with cardiac pacemakers and underwent right ventricular pacing to induce heart failure. Cardiac structure and function were evaluated. The gene expression and protein level of ICl,stretch in the left atrial appendage were detected. RESULTS: The left atrial diameter, right atrial dimension, left ventricular diastolic dimension, and right ventricular diastolic dimension were significantly larger in the heart failure group (P < 0.05). In contrast, the ejection fraction and the left ventricular shorten fraction were higher in the control group (P < 0.05). Both the mRNA and protein expression levels of ICl,stretch in atrial myocardium of the heart failure group were significantly higher compared with the control group. CONCLUSION: ICl,stretch might play an important role in the vulnerability to atrial fibrillation in dilated atria with heart failure and could be a potential therapeutic target for atrial fibrillation.
Asunto(s)
Fibrilación Atrial/genética , Regulación de la Expresión Génica , Atrios Cardíacos/metabolismo , Insuficiencia Cardíaca/complicaciones , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Miocardio/metabolismo , Volumen Sistólico/fisiología , Animales , Fibrilación Atrial/etiología , Fibrilación Atrial/metabolismo , Modelos Animales de Enfermedad , Perros , Femenino , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Masculino , ARN/genéticaRESUMEN
The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory ß1-subunits. Although the short cytosolic N-terminal domain of the ß1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the ß1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the ß1-subunit N terminus.
Asunto(s)
Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/biosíntesis , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Dominios Proteicos , Transporte de Proteínas/fisiologíaRESUMEN
BACKGROUND: Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA-FLS) contribute to joint inflammation and damage characteristic of the disease. RA-FLS express KCa1.1 (BK, Slo1, MaxiK, KCNMA1) as their major plasma membrane potassium channel. Blocking KCa1.1 reduces the invasive phenotype of RA-FLS and attenuates disease severity in animal models of RA. This channel has therefore emerged as a promising therapeutic target in RA. However, the pore-forming α subunit of KCa1.1 is widely distributed in the body, and blocking it induces severe side effects, thus limiting its value as a therapeutic target. On the other hand, KCa1.1 channels can also contain different accessory subunits with restricted tissue distribution that regulate channel kinetics and pharmacology. Identification of the regulatory subunits of KCa1.1 expressed by RA-FLS may therefore provide the opportunity for generating a selective target for RA treatment. METHODS: Highly invasive RA-FLS were isolated from patients with RA, and FLS from patients with osteoarthritis (OA) were used as minimally invasive controls. The ß subunit expression by FLS was assessed by quantitative reverse transcription polymerase chain reactions, Western blotting, and patch-clamp electrophysiology combined with pharmacological agents. FLS were sorted by flow cytometry on the basis of their CD44 expression level for comparison of their invasiveness and with their expression of KCa1.1 α and ß subunits. ß1 and ß3 subunit expression was reduced with small interfering RNA (siRNA) to assess their specific role in KCa1.1α expression and function and in FLS invasiveness. RESULTS: We identified functional ß1 and ß3b regulatory subunits in RA-FLS. KCa1.1 ß3b subunits were expressed by 70 % of the cells and were associated with highly invasive CD44(high) RA-FLS, whereas minimally invasive CD44(low) RA-FLS and OA-FLS expressed either ß1 subunit. Furthermore, we found that silencing the ß3 but not the ß1 subunit with siRNA reduced KCa1.1 channel density at the plasma membrane of RA-FLS and inhibited RA-FLS invasiveness. CONCLUSIONS: Our findings suggest the KCa1.1 channel composed of α and ß3b subunits as an attractive target for the therapy of RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Sinoviocitos/metabolismo , Adulto , Anciano , Artritis Reumatoide/patología , Western Blotting , Movimiento Celular/fisiología , Femenino , Fibroblastos/patología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , Sinoviocitos/patologíaRESUMEN
Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-α (ER-α) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-α gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-α gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O2 and 10.5% O2 for 48 hours. Hypoxia significantly increased ER-α promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-α expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2'-deoxycytidine. In addition, hypoxia abrogated steroid hormone-mediated increase in ER-α expression and inhibited the hormone-induced increase in large-conductance Ca(2+)-activated K(+) channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2'-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-α gene repression and ablation of steroid hormone-mediated adaptation of uterine arterial large conductance Ca(2+)-activated K(+) channel activity and myogenic tone in pregnancy.
Asunto(s)
Metilación de ADN , Receptor alfa de Estrógeno/biosíntesis , Regulación de la Expresión Génica/genética , Hipoxia/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Complicaciones del Embarazo/genética , Arteria Uterina/metabolismo , Acetilcisteína/farmacología , Animales , Azacitidina/análogos & derivados , Azacitidina/farmacología , Metilación de ADN/efectos de los fármacos , Decitabina , Estradiol/farmacología , Estradiol/fisiología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/fisiología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipoxia/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Embarazo , Complicaciones del Embarazo/metabolismo , Progesterona/farmacología , Regiones Promotoras Genéticas , Especies Reactivas de Oxígeno , Ovinos , Arteria Uterina/efectos de los fármacos , Resistencia VascularRESUMEN
Cancer stem cells (CSCs) are usually tolerant to chemotherapy and radiotherapy and associated with tumor relapse. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), is currently being used in clinical trials of lung cancer. However, SAHA facilitates the formation of induced pluripotent stem cells from somatic cells. We hypothesized that SAHA would mediate the CSCs properties and subsequently confer a more malignant phenotype in lung cancer. Transfected H1299 lung cancer cells, which stably expresses a triple fused reporter gene (DsRedm-Fluc-tTKsr39) under the control of CMV promoter was used to establish a xenograft mouse model. After the treatment of SAHA, H1299 cell line and tumor xenografts were sorted by fluorescence-activated cell sorting (FACS) based on aldehyde dehydrogenase (ALDH) activity. We found that SAHA could suppress the growth of xenografted H1299 tumors with decreased proportion of ALDHbr lung cancer cells indicating that SAHA may target CSCs. However, SAHA significantly enhanced the tumor initiating capacity and the expression of malignant genes such as KCNMA1, MORF4L2 and ASPM in the remaining living ALDHbr cells. These findings suggested that SAHA treatment created a more drug-resistant state in residual ALDHbr cells. The in vivo imaging technique may facilitate searching and characterization of CSCs.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas de Neoplasias/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Factores de Transcripción/biosíntesis , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Aldehído Deshidrogenasa/análisis , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Autorrenovación de las Células/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Citometría de Flujo , Genes Reporteros , Xenoinjertos , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ácidos Hidroxámicos/uso terapéutico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Proteínas del Tejido Nervioso/genética , Factores de Transcripción/genética , Transfección , VorinostatRESUMEN
The metabolic activity of articular chondrocytes is influenced by osmotic alterations that occur in articular cartilage secondary to mechanical load. The mechanisms that sense and transduce mechanical signals from cell swelling and initiate volume regulation are poorly understood. The purpose of this study was to investigate how the expression of two putative osmolyte channels [transient receptor potential vanilloid 4 (TRPV4) and large-conductance Ca(2+)-activated K(+) (BKCa)] in chondrocytes is modulated in different osmotic conditions and to examine a potential role for MAPKs in this process. Isolated equine articular chondrocytes were subjected to anisosmotic conditions, and TRPV4 and BKCa channel expression and ERK1/2 and p38 MAPK protein phosphorylation were investigated using Western blotting. Results indicate that the TRPV4 channel contributes to the early stages of hypo-osmotic stress, while the BKCa channel is involved in responding to elevated intracellular Ca(2+) and mediating regulatory volume decrease. ERK1/2 is phosphorylated by hypo-osmotic stress (P < 0.001), and p38 MAPK is phosphorylated by hyperosmotic stress (P < 0.001). In addition, this study demonstrates the importance of endogenous ERK1/2 phosphorylation in TRPV4 channel expression, where blocking ERK1/2 by a specific inhibitor (PD98059) prevented increased levels of the TRPV4 channel in cells exposed to hypo-osmotic stress and decreased TRPV4 channel expression to below control levels in iso-osmotic conditions (P < 0.001).
Asunto(s)
Condrocitos/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Presión Osmótica/fisiología , Canales Catiónicos TRPV/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Calcio/metabolismo , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Condrocitos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica , Caballos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Presión Osmótica/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
This study was designed to determine the effects of aerobic exercise training on aging-associated selective changes of the function and expression of the large-conductance Ca(2+)-activated K(+) (MaxiK) channels in mesenteric arteries. Male Wistar rats aged 19-21 months were randomly assigned to sedentary (O-SED) and exercise-trained groups (O-EX). Two-month-old rats were used as Young control. Addition of iberiotoxin (10(-8) M) increased the norepinephrine-induced arterial contraction in all three groups, with the greatest enhancement being in Young and the least in O-SED. Patch clamp study revealed the characteristics of aging on MaxiK channel function in mesenteric arteries, mainly including (a) decrease of iberiotoxin-sensitive whole-cell K(+) current, (b) decrease of open probability and Ca(2+)/voltage sensitivity of single MaxiK channel, and (c) reduction of tamoxifen-induced MaxiK activation. After exercise training, all of these changes were markedly inhibited. Western blotting revealed that the protein expression of MaxiK was significantly reduced with aging and the suppression of ß1-subunit was larger than that of α-subunit, although exercise training diminished this alteration. Taken together, aerobic exercise training reverses the aging-related unparallel downregulation of MaxiK α- and ß1-subunit expression on mesenteric arteries, which partly underlies the beneficial effect of exercise on restoring aging-associated reduction in mesenteric artery vasodilatory properties.
Asunto(s)
Envejecimiento/fisiología , Regulación hacia Abajo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/biosíntesis , Arterias Mesentéricas/fisiología , Condicionamiento Físico Animal/fisiología , Vasoconstricción/fisiología , Animales , Western Blotting , Masculino , Músculo Liso Vascular/fisiología , Ratas , Ratas WistarRESUMEN
Large conductance calcium-activated potassium (BKCa) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BKCa channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BKCa channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BKCa channel activity in sensory neurons with particular pain modalities, we generated mice in which BKCa channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BKCa channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BKCa channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states.
Asunto(s)
Regulación de la Expresión Génica , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Dimensión del Dolor/métodos , Dolor/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Femenino , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Dolor/patología , Células Receptoras Sensoriales/patologíaRESUMEN
Airway smooth muscle (ASM) cell phenotype modulation, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to proliferative diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD). KCa3.1 has been suggested to be involved in regulating ASM cell activation, proliferation, and migration. However, little is known regarding the exact role of KCa3.1 in ASM cell phenotypic modulation. To elucidate the role of KCa3.1 in regulating ASM cell phenotypic modulation, we investigated the effects of KCa3.1 channels on ASM contractile marker protein expression, proliferation and migration of primary human bronchial smooth muscle (BSM) cells. We found that PDGF increased KCa3.1 channel expression in BSM cells with a concomitant marked decrease in the expression of contractile phenotypic marker proteins including smooth muscle myosin heavy chain (SMMHC), smooth muscle α-actin (α-SMA), myocardin and KCa1.1. These changes were significantly attenuated by the KCa3.1 blocker, TRAM-34, or gene silencing of KCa3.1. Pharmacological blockade or gene silencing of KCa3.1 also suppressed PDGF-induced human BSM cell migration and proliferation accompanied by a decrease in intracellular free Ca(2+) levels as a consequence of membrane depolarization, resulting in a reduction in cyclin D1 level and cell cycle arrest at G0-G1 phase. Additionally, PDGF-induced up-regulation of KCa3.1 and down-regulation of BSM contractile marker proteins were regulated by the ERK inhibitor U0126 and the AKT inhibitor LY294002. These findings highlight a novel role for the KCa3.1 channel in human BSM cell phenotypic modulation and provide a potential target for therapeutic intervention for proliferative airway diseases.
Asunto(s)
Canales de Potasio de Conductancia Intermedia Activados por el Calcio/biosíntesis , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/fisiología , Miocitos del Músculo Liso/fisiología , Fenotipo , Regulación hacia Arriba , Actinas/biosíntesis , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Bronquios/fisiología , Butadienos/farmacología , Calcio/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Ciclina D1/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Silenciador del Gen , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Morfolinas/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/biosíntesis , Nitrilos/farmacología , Proteínas Nucleares/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Transactivadores/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Defective colonic Na(+) and Cl(-) absorption is a feature of active ulcerative colitis (UC), but little is known about changes in colonic K(+) transport. We therefore investigated colonic K(+) transport in a rat model of dextran sulfate-induced colitis. Colitis was induced in rat distal colon using 5% dextran sulfate sodium (DSS). Short-circuit current (Isc, indicating electrogenic ion transport) and (86)Rb (K(+) surrogate) fluxes were measured in colonic mucosa mounted in Ussing chambers under voltage-clamp conditions in the presence of mucosal orthovanadate (a P-type ATPase inhibitor). Serum aldosterone was measured by immunoassay. Control animals exhibited zero net K(+) flux. By contrast, DSS-treated animals exhibited active K(+) secretion, which was inhibited by 98, 76, and 22% by Ba(2+) (nonspecific K(+) channel blocker), iberiotoxin (IbTX; BK channel blocker), and TRAM-34 (IK channel blocker), respectively. Apical BK channel α-subunit mRNA abundance and protein expression, and serum aldosterone levels in DSS-treated animals, were enhanced 6-, 3-, and 6-fold respectively, compared with controls. Increasing intracellular Ca(2+) with carbachol (CCH), or intracellular cAMP with forskolin (FSK), stimulated both active Cl(-) secretion and active K(+) secretion in controls but had no or little effect in DSS-treated animals. In DSS-induced colitis, active K(+) secretion involves upregulation of apical BK channel expression, which may be aldosterone-dependent, whereas Cl(-) secretion is diminished. Since similar ion transport abnormalities occur in patients with UC, diarrhea in this disease may reflect increased colonic K(+) secretion (rather than increased Cl(-) secretion), as well as defective Na(+) and Cl(-) absorption.
Asunto(s)
Colitis/metabolismo , Sulfato de Dextran/toxicidad , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Potasio/metabolismo , Regulación hacia Arriba/fisiología , Animales , Colitis/inducido químicamente , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Masculino , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/efectos de los fármacosRESUMEN
MicroRNAs (miR) regulate phenotype and function of neurons by binding to miR-response elements (MRE) in the 3' untranslated regions (3'UTR) of various messenger RNAs to inhibit translation. MiR expression can be induced or inhibited by environmental factors like drug exposure and viral infection, leading to changes in cellular physiology. We hypothesized that the effects of methamphetamine (MA) and human immunodeficiency virus (HIV)-infection in the brain will induce changes in miR expression, and have downstream regulatory consequences in neurons. We first used a PCR-based array to screen for differential expression of 380 miRs in frontal cortex autopsy tissues of HIV-positive MA abusers and matched controls. These results showed significantly increased expression of the neuron-specific miR-9. In vitro, we used SH-SY5Y cells, an experimental system for dopaminergic studies, to determine miR expression by quantitative PCR after exposure to MA in the presence or absence of conditioned media from HIV-infected macrophages. Again, we found that miR-9 was significantly increased compared to controls. We also examined the inwardly rectifying potassium channel, KCNMA1, which has alternative splice variants that contain an MRE to miR-9. We identified alternate 3'UTRs of KCNMA1 both in vitro and in the autopsy specimens and found differential splice variant expression of KCNMA1, operating via the increased miR-9. Our results suggest that HIV and MA -induced elevated miR-9, leading to suppression of MRE-containing splice variants of KCNMA1, which may affect neurotransmitter release in dopaminergic neurons.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Estimulantes del Sistema Nervioso Central/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Metanfetamina/farmacología , MicroARNs/biosíntesis , Neuronas/virología , Complejo SIDA Demencia/genética , Adulto , Autopsia , Estudios de Casos y Controles , Línea Celular , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Hibridación in Situ , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Isoformas de Proteínas , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Large-conductance Ca(2+)-activated K(+) channels (BKCa) are widely expressed in the central nervous system and play important roles in neural activities. Nicotine exposure leads to long-lasting changes in behavioral and neuronal plasticity. However, little is known the roles of BKCa in the development of nicotine addiction. In the present study, a significant reduction in BKCa channel expression was found in nucleus accumbens (NAc) from nicotine addiction mice. Whole-cell patch-clamp recordings from NAc neurons of the addicted animals revealed a pronounced reduction in the fast after-hyperpolarization of action potentials mediated by BKCa channels that led to hyperexcitability of the NAc neurons. Activation of BKCa channels in the NAc reversed drug-seeking behaviors which were detected by conditioned place preference test. Furthermore, knockdown of BKCa channels using short hairpin RNAs significantly increased the drug-seeking behavior. These findings provide direct evidence that alterations of BKCa channels in the NAc play critical roles in the development of nicotine addiction and that modulation of the BKCa channels may be potential therapeutics for drug addiction.
Asunto(s)
Comportamiento de Búsqueda de Drogas/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Proteínas del Tejido Nervioso/metabolismo , Nicotina/farmacología , Núcleo Accumbens/metabolismo , Tabaquismo/metabolismo , Potenciales de Acción , Animales , Calcio/fisiología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Condicionamiento Clásico/efectos de los fármacos , Regulación hacia Abajo , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Conducta Exploratoria/efectos de los fármacos , Regulación de la Expresión Génica , Transporte Iónico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología , Nicotina/administración & dosificación , Núcleo Accumbens/fisiopatología , Técnicas de Placa-Clamp , Potasio/metabolismo , ARN Interferente Pequeño/farmacología , Recompensa , Tabaquismo/genéticaRESUMEN
We determined the possible role of large-conductance Ca2+-activated K (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel α- and ß1-subunits were coexpressed, unitary BK currents were detected, and single-channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca sparks and iberiotoxin-sensitive spontaneous transient outward currents. Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-induced, KCl-induced (20 mM), and BayK8644-induced contraction. Acute inhibition of BK channels with iberiotoxin in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel α-subunits and L-type Ca2+ channel α1-C subunits are expressed in murine MV. However, these channels are not functional because murine MV lack nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl, and BayK8644, consistent with our previous studies showing that murine MV do not have BK ß1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone but also BK channels are required for rhythmic oscillations in venous tone.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Venas Mesentéricas/metabolismo , Contracción Muscular/fisiología , Músculo Liso Vascular/metabolismo , Vasodilatación/fisiología , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Células Cultivadas , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/biosíntesis , Masculino , Venas Mesentéricas/efectos de los fármacos , Venas Mesentéricas/fisiopatología , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Vasodilatación/efectos de los fármacosRESUMEN
Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.
Asunto(s)
Membrana Celular/metabolismo , Regulación de la Expresión Génica , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Fiebre Reumática/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Calcio/metabolismo , Membrana Celular/patología , Proliferación Celular , Precursores Enzimáticos/biosíntesis , Femenino , Gelatinasas/biosíntesis , Células HEK293 , Homeostasis , Humanos , Interleucina-8/biosíntesis , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Fiebre Reumática/patología , Membrana Sinovial/patología , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K(+) channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. The glutathione S-transferase pull-down assay demonstrated that MAGI-1 was a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na(+) -dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1 mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/biosíntesis , Guanilato-Quinasas/biosíntesis , Proteínas de la Membrana/biosíntesis , Animales , Astrocitos/metabolismo , Biotinilación , Línea Celular Tumoral , Células Cultivadas , Transportador 1 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/biosíntesis , Transportador 3 de Aminoácidos Excitadores/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Glioma/metabolismo , Ácido Glutámico/metabolismo , Glutatión Transferasa/metabolismo , Guanilato-Quinasas/genética , Hipocampo/citología , Hipocampo/metabolismo , Inmunoprecipitación , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Proteínas de la Membrana/genética , Plásmidos/genética , ARN Interferente Pequeño/farmacología , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , TransfecciónRESUMEN
BACKGROUND AND PURPOSE: Bisphenol A (BPA) is used to manufacture plastics, including containers for food into which it may leach. High levels of exposure to this oestrogenic endocrine disruptor are associated with diabetes and heart disease. Oestrogen and oestrogen receptor modulators increase the activity of large conductance Ca(2+)/voltage-sensitive K(+) (Maxi-K; K(Ca)1.1) channels, but the effects of BPA on Maxi-K channels are unknown. We tested the hypothesis that BPA activates Maxi-K channels through a mechanism that depends upon the regulatory beta1 subunit. EXPERIMENTAL APPROACH: Patch-clamp recordings of Maxi-K channels were made in human and canine coronary smooth muscle cells as well as in AD-293 cells expressing pore-forming alpha or alpha plus beta1 subunits. KEY RESULTS: BPA (10 microM) activated an outward current in smooth muscle cells that was inhibited by penitrem A (1 microM), a Maxi-K blocker. BPA increased Maxi-K activity in inside-out patches from coronary smooth muscle, but had no effect on single channel conductance. In AD-293 cells with Maxi-K channels composed of alpha subunits alone, 10 microM BPA did not affect channel activity. When channels in AD-293 cells contained beta1 subunits, 10 microM BPA increased channel activity. Effects of BPA were rapid (<1 min) and reversible. A higher concentration of BPA (100 microM) increased Maxi-K current independent of the beta1 subunit. CONCLUSIONS AND IMPLICATIONS: Our data indicate that BPA increased the activity of Maxi-K channels and may represent a basis for some potential toxicological effects.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Disruptores Endocrinos/toxicidad , Canales de Potasio de Gran Conductancia Activados por el Calcio/agonistas , Miocitos del Músculo Liso/efectos de los fármacos , Fenoles/toxicidad , Animales , Compuestos de Bencidrilo , Células Cultivadas , Vasos Coronarios/citología , Vasos Coronarios/fisiología , Perros , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/agonistas , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/agonistas , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/biosíntesis , Canales de Potasio de Gran Conductancia Activados por el Calcio/biosíntesis , Miocitos del Músculo Liso/fisiología , Técnicas de Placa-ClampRESUMEN
Pharmacological activation of the large-conductance Ca(2+)-activated K(+) channel (KCa1.1) in the cardiac inner mitochondrial membrane has been found to protect the heart against ischemia reperfusion injuries. However, there are concerns about the selectivity of the pharmacological tools used to modulate the channel. Here, we address this issue by synthesising a methylated analogue of the tool KCa1.1 channel activator NS11021. The compound (NS13558) is designed as a structurally closely related and biologically inactive analogue of NS11021. NS13558 did not elicit any significant opening of cloned human KCa1.1 channels, but maintained comparable biological activity towards other cardiac ion channels as compared to NS11021. In isolated perfused rat hearts subjected to ischemia-reperfusion, infarct size was reduced from 29% in control to 7% in NS11021 treated hearts. In comparison, the inactive derivate of NS11021, i.e., NS13558, did not confer any cardioprotection, demonstrated by an infarct size identical to control hearts. This suggests that NS11021 exerts its primary effect through KCa1.1 channels, which indicates an important role of these channels in protection against ischemia-reperfusion injuries. Furthermore, the study demonstrates a novel way of combining an activator of the KCa1.1 channel (NS11021) and its structurally closely related inactive analogue NS13558 to address the functional role of KCa1.1 channels, and we believe these novel tools may constitute a valuable addition to understanding the functional role of KCa1.1 channels under physiological and pathophysiological conditions.
Asunto(s)
Cardiotónicos/síntesis química , Cardiotónicos/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/agonistas , Tetrazoles/síntesis química , Tetrazoles/farmacología , Tiourea/análogos & derivados , Potenciales de Acción/efectos de los fármacos , Animales , Cardiotónicos/uso terapéutico , Línea Celular , Humanos , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Masculino , Estructura Molecular , Infarto del Miocardio/etiología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Perfusión , Ratas , Ratas Sprague-Dawley , Tetrazoles/química , Tetrazoles/uso terapéutico , Tiourea/química , Tiourea/farmacología , Tiourea/uso terapéutico , Transfección , Xenopus laevisRESUMEN
We carried out a yeast two-hybrid screen to identify proteins that interact with large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene. Nephrin, an essential adhesion and scaffolding molecule expressed in podocytes, emerged in this screen. The Slo1-nephrin interaction was confirmed by coimmunoprecipitation from the brain and kidney, from HEK-293T cells expressing both proteins, and by glutathione S-transferase pull-down assays. We detected nephrin binding to the Slo1 VEDEC splice variant, which is typically retained in intracellular stores, and to the beta4-subunit. However, we did not detect significant binding of nephrin to the Slo1 QEERL or Slo1 EMVYR splice variants. Coexpression of nephrin with Slo1 VEDEC increased expression of functional BKCa channels on the surface of HEK-293T cells but did not affect steady-state surface expression of the other COOH-terminal Slo1 variants. Nephrin did not affect the kinetics or voltage dependence of channel activation in HEK-293T cells expressing Slo1. Stimulation of Slo1 VEDEC surface expression in HEK-293T cells was also observed by coexpressing a small construct encoding only the distal COOH-terminal domains of nephrin that interact with Slo1. Reduction of endogenous nephrin expression by application of small interfering RNA to differentiated cells of an immortalized podocyte cell line markedly reduced the steady-state surface expression of Slo1 as assessed by electrophysiology and cell-surface biotinylation assays. Nephrin therefore plays a role in organizing the surface expression of ion channel proteins in podocytes and may play a role in outside-in signaling to allow podocytes to adapt to mechanical or neurohumoral stimuli originating in neighboring cells.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Pollos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Riñón/embriología , Riñón/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Isoformas de Proteínas/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels regulate the physiological properties of many cell types. The gating properties of BK(Ca) channels are Ca(2+)-, voltage- and stretch-sensitive, and stretch-sensitive gating of these channels requires interactions with actin microfilaments subjacent to the plasma membrane. Moreover, we have previously shown that trafficking of BK(Ca) channels to the plasma membrane is associated with processes that alter cytoskeletal dynamics. Here, we show that the Slo1 subunits of BK(Ca) channels contain a novel cytoplasmic actin-binding domain (ABD) close to the C terminus, considerably downstream from regions of the channel molecule that play a major role in determining channel-gating properties. Binding of actin to the ABD can occur in a binary mixture in the absence of other proteins. Coexpression of a small ABD-green fluorescent protein fusion protein that competes with full-length Slo1 channels for binding to actin markedly suppresses trafficking of full-length Slo1 channels to the plasma membrane. In addition, Slo1 channels containing deletions of the ABD that eliminate actin binding are retained in intracellular pools, and they are not expressed on the cell surface. At least one point mutation within the ABD (L1020A) reduces surface expression of Slo1 channels to approximately 25% of wild type, but it does not cause a marked effect on the gating of point mutant channels that reach the cell surface. These data suggest that Slo1-actin interactions are necessary for normal trafficking of BK(Ca) channels to the plasma membrane and that the mechanisms of this interaction may be different from those that underlie F-actin and stretch-sensitive gating.
Asunto(s)
Actinas/biosíntesis , Membrana Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/biosíntesis , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Actinas/genética , Sitios de Unión/fisiología , Línea Celular , Membrana Celular/genética , Células Cultivadas , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Canales de Potasio Calcio-Activados/biosíntesis , Canales de Potasio Calcio-Activados/genética , Unión Proteica/fisiología , Estructura Terciaria de Proteína/genéticaRESUMEN
Riluzole is known to be of therapeutic use in the management of amyotrophic lateral sclerosis. In this study, we investigated the effects of riluzole on ion currents in cultured differentiated human skeletal muscle cells (dHSkMCs). Western blotting revealed the protein expression of alpha-subunits for both large-conductance Ca2+-activated K+ (BK(Ca)) channel and Na+ channel (Na(v)1.5) in these cells. Riluzole could reduce the frequency of spontaneous beating in dHSkMCs. In whole-cell configuration, riluzole suppressed voltage-gated Na+ current (I(Na)) in a concentration-dependent manner with an IC50 value of 2.3 microM. Riluzole (10 microM) also effectively increased Ca2+-activated K+ current (I(K(Ca))) which could be reversed by iberiotoxin (200 nM) and paxilline (1 microM), but not by apamin (200 nM). In inside-out patches, when applied to the inside of the cell membrane, riluzole (10 microM) increased BK(Ca)-channel activity with a decrease in mean closed time. Simulation studies also unraveled that both decreased conductance of I(Na) and increased conductance of I(K(Ca)) utilized to mimic riluzole actions in skeletal muscle cells could combine to decrease the amplitude of action potentials and increase the repolarization of action potentials. Taken together, inhibition of I(Na) and stimulation of BK(Ca)-channel activity caused by this drug are partly, if not entirely, responsible for its muscle relaxant actions in clinical setting.