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1.
J Biol Chem ; 287(40): 33132-41, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22879586

RESUMO

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (I(Kr)) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native I(Kr). Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology.


Assuntos
Caveolina 3/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais de Potássio Éter-A-Go-Go/metabolismo , Regulação da Expressão Gênica , Ubiquitina-Proteína Ligases/metabolismo , Animais , Membrana Celular/metabolismo , Feminino , Células HEK293 , Ventrículos do Coração/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Células Musculares/citologia , Ubiquitina-Proteína Ligases Nedd4 , Técnicas de Patch-Clamp , Mutação Puntual , RNA Interferente Pequeno/metabolismo , Ratos
2.
Mol Pharmacol ; 79(5): 806-13, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21278233

RESUMO

The human ether-à-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier K(+) current (I(Kr)) important for cardiac repolarization. Dysfunction of the hERG channel causes long QT syndrome (LQTS). Although diverse compounds reduce the hERG current (I(hERG)) by blocking the channel, probucol, a cholesterol-lowering drug that causes LQTS, reduces I(hERG) by decreasing plasma-membrane hERG protein expression. Here, we investigated the mechanisms of probucol effects on hERG expression levels. Our data demonstrate that probucol accelerated the degradation of mature hERG channels, which associated with caveolin-1 (Cav1) in hERG-expressing HEK cells. In human embryonic kidney (HEK) cells without hERG expression, probucol promoted endogenous Cav1 degradation. In hERG-expressing HEK cells, overexpression of Cav1 enhanced, whereas knockdown of Cav1 impeded, probucol-induced reduction of mature hERG channels. Thus, probucol reduces hERG expression through accelerating Cav1 turnover. The effects of probucol on Cav1 and hERG result from probucol's cholesterol-disrupting action, because low-density lipoprotein (LDL), a potent cholesterol carrier, effectively prevented probucol-induced reduction of I(hERG) in hERG-expressing HEK cells and of I(Kr) in neonatal rat cardiomyocytes. Our data provide evidence that targeting hERG-interacting protein caveolin represents a novel mechanism for drugs to decrease hERG expression and cause LQTS.


Assuntos
Anticolesterolemiantes/farmacologia , Caveolina 1/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Síndrome do QT Longo/etiologia , Proteínas de Membrana/efeitos dos fármacos , Probucol/farmacologia , Linhagem Celular , Canal de Potássio ERG1 , Humanos , Hidrólise , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Técnicas de Patch-Clamp
3.
J Biol Chem ; 286(8): 6751-9, 2011 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-21177251

RESUMO

A reduction in extracellular K(+) concentration ([K(+)](o)) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (I(Kr)) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K(+) conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K(+)-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K(+) conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K(+) conditions.


Assuntos
Endocitose/fisiologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Lisossomos/metabolismo , Potássio/farmacologia , Ubiquitinação/fisiologia , Canal de Potássio ERG1 , Endocitose/efeitos dos fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Lisossomos/genética , Mutação , Potássio/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos
4.
J Biol Chem ; 285(35): 27259-27264, 2010 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-20605793

RESUMO

Reduction in the rapidly activating delayed rectifier K(+) channel current (I(Kr)) due to either mutations in the human ether-a-go-go-related gene (hERG) or drug block causes inherited or drug-induced long QT syndrome. A reduction in extracellular K(+) concentration ([K(+)](o)) exacerbates long QT syndrome. Recently, we demonstrated that lowering [K(+)](o) promotes degradation of I(Kr) in rabbit ventricular myocytes and of the hERG channel stably expressed in HEK 293 cells. In this study, we investigated the degradation pathways of hERG channels under low K(+) conditions. We demonstrate that under low K(+) conditions, mature hERG channels and caveolin-1 (Cav1) displayed a parallel time-dependent reduction. Mature hERG channels coprecipitated with Cav1 in co-immunoprecipitation analysis, and internalized hERG channels colocalized with Cav1 in immunocytochemistry analysis. Overexpression of Cav1 accelerated internalization of mature hERG channels in 0 mM K(+)(o), whereas knockdown of Cav1 impeded this process. In addition, knockdown of dynamin 2 using siRNA transfection significantly impeded hERG internalization and degradation under low K(+)(o) conditions. In cultured neonatal rat ventricular myocytes, knockdown of caveolin-3 significantly impeded low K(+)(o)-induced reduction of I(Kr). Our data indicate that a caveolin-dependent endocytic route is involved in low K(+)(o)-induced degradation of mature hERG channels.


Assuntos
Caveolina 1/metabolismo , Endocitose , Canais de Potássio Éter-A-Go-Go/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Animais , Caveolina 1/genética , Linhagem Celular , Dinamina II/genética , Dinamina II/metabolismo , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Humanos , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Masculino , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley
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