RESUMEN
The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.
Asunto(s)
Canal de Potasio Kv.1.1/metabolismo , Canal de Potasio Kv1.3/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Proteolípidos/efectos de los fármacos , Venenos Elapídicos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Isoxazoles/química , Canal de Potasio Kv.1.1/genética , Canal de Potasio Kv1.3/genética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/metabolismo , Valinomicina/farmacologíaRESUMEN
BACKGROUND/AIMS: Thiazolidinediones (TZDs), such as rosiglitazone or pioglitazone, are peroxisome proliferator-activated receptor gamma (PPARγ) agonists currently used in the treatment of type 2 diabetes. However, their clinical applicability is limited by common and severe side effects including strong water retention, edema and cardiac stroke. The precise mechanisms leading to these disorders are not clearly understood and remain controversial. While the nature of the disorders due to TZDs points to an increase in ENaC-mediated sodium reabsorption in the aldosterone-sensitive distal nephron, some studies suggested that this channel was not targeted by PPARγ agonists. METHODS: Mouse cortical collecting duct cells were incubated in different types of culture medium and treated with or without rosiglitazone. Transepithelial Na(+) current was measured and the changes in SGK and Nedd4 expression were determined by immunoblotting. RESULTS: Herein we demonstrate that rosiglitazone stimulates the amiloride-sensitive transepithelial sodium current in Collecting Duct Principal Cells after 3h and 24h treatment. This activation was dependent of both serum and insulin in culture medium and was mediated by SGK1/Nedd4-2 pathway stimulation. In these conditions, rosiglitazone induced SGK1 expression, Nedd4-2 phosphorylation and thus abolished ubiquitylation and internalization of ENaC channels. This mechanism explains most of the side effects of thiazolidinediones previously observed in humans and animals. CONCLUSION: Our data show an increase in transepithelial sodium amiloride-sensitive current induced by a PPARγ agonist in presence of serum and insulin, thus confirming some in-vitro and in-vivo experiments while providing explanations for previous conflicting findings.
Asunto(s)
Canales Iónicos Sensibles al Ácido/metabolismo , Canales Epiteliales de Sodio/metabolismo , Insulina/farmacología , Túbulos Renales Colectores/metabolismo , PPAR gamma/metabolismo , Suero , Tiazolidinedionas/farmacología , Animales , Línea Celular Transformada , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Ubiquitina-Proteína Ligasas Nedd4 , PPAR gamma/agonistas , Proteínas Serina-Treonina Quinasas/metabolismo , Rosiglitazona , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Activación del Canal Iónico/fisiología , Oocitos/fisiología , PPAR gamma/agonistas , PPAR gamma/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Tiazolidinedionas/farmacología , Animales , Células Cultivadas , Canales Epiteliales de Sodio/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Oocitos/efectos de los fármacos , Rosiglitazona , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estadística como Asunto , Regulación hacia Arriba/efectos de los fármacos , Xenopus laevisRESUMEN
The epithelial sodium channel (ENaC) is regulated by hormones and by other intracellular or extracellular factors. It is activated by the sulfonylurea drug glibenclamide. The activator effect of glibenclamide is fast and reversible and was observed in Xenopus oocytes coexpressing the alpha subunit from human, Xenopus, or guinea pig (but not rat) with betagamma-rat ENaC subunits. The mechanism of this effect is not yet well understood. We hypothesize that the extracellular loop of ENaC plays a major role in this activation. Mutants and chimeras of alpha subunits harboring different parts of the rat and guinea pig alpha-subunit extracellular loops were generated and coexpressed with betagamma-rat subunits in Xenopus oocytes. The effect of glibenclamide on ENaC activity was measured using two-electrode voltage-clamp technique. The alpha-rat ENaC chimera containing the C-terminal part of the extracellular loop of the alpha-guinea pig ENaC was significantly stimulated by glibenclamide (1.26-fold), whereas the rat-alpha combination was not activated by this sulfonylurea. Mutagenesis of specific residues on the rat alpha subunit did not generate channels sensitive to glibenclamide, suggesting that the overall structure of the extracellular loop is required for activation of the channel by this drug. These results support the hypothesis of the existence of a role played by the last 100 amino acids of the extracellular loop and confirm that the ENaC behaves as a ligand-gated channel similar to several other members of the ENaC/degenerin family.
Asunto(s)
Agonistas del Canal de Sodio Epitelial , Canales Epiteliales de Sodio/química , Gliburida/farmacología , Hipoglucemiantes/farmacología , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Electrofisiología , Canales Epiteliales de Sodio/genética , Cobayas , Humanos , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Ratas , Homología de Secuencia de Aminoácido , Bloqueadores de los Canales de Sodio/farmacología , XenopusRESUMEN
The epithelial sodium channel (ENaC) is the major rate-limiting step for vasopressin and aldosterone sensitive Na(+) reabsorption across kidney epithelia. Recently, ENaC activity was shown to be modulated by extracellular factors such as proteases, Na(+) ion and several other elements. However, the molecular mechanisms of these actions remain unclear. We and others have shown that ENaC composed of the guinea-pig alpha-subunit (alphagp), and the beta gamma rat subunits (betargammar) could be activated by cpt-cAMP, a cAMP analogue, through a mechanism not involving the cAMP-PKA pathway. In the present study, we confirmed by patch-clamp experiments on Xenopus oocytes that the number of open channels increased by 2.4-fold after cpt-cAMP exposure. In order to characterize the extracellular domain involved in this activation, we generated alpha-subunit chimera's harboring different portions of the extracellular loop of the alphagp and alphar. Using two-electrode voltage-clamp, we established that Tyr456-Ser532 from the alphagp confers sensibility to cpt-AMP. Then, by site-directed mutagenesis, we have isolated Ile481 as a major residue for cpt-cAMP-dependant activation. Taken together, these experiments provide evidence of an extracellular-ligand stimulating ENaC. They also contribute to the further understanding of the structure-function relationship of this channel.