RESUMEN
Two recent studies (Newton-Cheh, C. et al. (2009) Common variants at ten loci influence QT interval duration in the QTGEN Study. Nat. Genet. 41, 399-406 and Pfeufer, A. et al. (2009) Common variants at ten loci modulate the QT interval duration in the QTSCD Study. Nat. Genet. 41, 407-414) identified an association, with genome-wide significance, between a single nucleotide polymorphism within the gene encoding RING finger protein 207 (RNF207) and the QT interval. We sought to determine the role of RNF207 in cardiac electrophysiology. Morpholino knockdown of RNF207 in zebrafish embryos resulted in action potential duration prolongation, occasionally a 2:1 atrioventricular block, and slowing of conduction velocity. Conversely, neonatal rabbit cardiomyocytes infected with RNF207-expressing adenovirus exhibited shortened action potential duration. Using transfections of U-2 OS and HEK293 cells, Western blot analysis and immunocytochemistry data demonstrate that RNF207 and the human ether-a-go-go-related gene (HERG) potassium channel interact and colocalize. Furthermore, RNF207 overexpression significantly elevated total and membrane HERG protein and HERG-encoded current density by â¼30-50%, which was dependent on the intact N-terminal RING domain of RNF207. Finally, coexpression of RNF207 and HSP70 increased HERG expression compared with HSP70 alone. This effect was dependent on the C terminus of RNF207. Taken together, the evidence is strong that RNF207 is an important regulator of action potential duration, likely via effects on HERG trafficking and localization in a heat shock protein-dependent manner.
Asunto(s)
Bloqueo Atrioventricular/genética , Canales de Potasio Éter-A-Go-Go/genética , Proteínas HSP70 de Choque Térmico/genética , Corazón/fisiología , Miocitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Potenciales de Acción/genética , Adenoviridae/genética , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Bloqueo Atrioventricular/metabolismo , Bloqueo Atrioventricular/fisiopatología , Canal de Potasio ERG1 , Embrión no Mamífero , Canales de Potasio Éter-A-Go-Go/metabolismo , Acoplamiento Excitación-Contracción , Regulación de la Expresión Génica , Vectores Genéticos , Células HEK293 , Proteínas HSP70 de Choque Térmico/metabolismo , Corazón/embriología , Corazón/fisiopatología , Humanos , Datos de Secuencia Molecular , Morfolinos , Miocitos Cardíacos/patología , Estructura Terciaria de Proteína , Conejos , Ubiquitina-Proteína Ligasas/metabolismo , Pez CebraRESUMEN
KCNQ1 and hERG encode the voltage-gated potassium channel α-subunits of the cardiac repolarizing currents I(Ks) and I(Kr), respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs), and loss-of-function mutations in these channels manifest clinically as long QT syndrome, characterized by the prolongation of the QT interval, polymorphic ventricular tachycardia, and sudden cardiac death. Previous cellular electrophysiology experiments in transgenic rabbit cardiomyocytes and heterologous cell lines demonstrated functional downregulation of complementary repolarizing currents. Biochemical assays indicated direct, protein-protein interactions between KCNQ1 and hERG may underlie the interplay between I(Ks) and I(Kr). Our objective was to investigate hERG-KCNQ1 interactions in the intact cellular environment primarily through acceptor photobleach FRET (apFRET) experiments. We quantitatively assessed the extent of interactions based on fluorophore location and the potential regulation of interactions by physiologically relevant signals. apFRET experiments established specific hERG-KCNQ1 associations in both heterologous and primary cardiomyocytes. The largest FRET efficiency (E(f); 12.0 ± 5.2%) was seen between ion channels with GFP variants fused to the COOH termini. Acute treatment with forskolin + IBMX or a membrane-permeable cAMP analog significantly and specifically reduced the extent of hERG-KCNQ1 interactions (by 41 and 38%, respectively). Our results demonstrate direct interactions between KCNQ1 and hERG occur in both intact heterologous cells and primary cardiomyocytes and are mediated by their COOH termini. Furthermore, this interplay between channel proteins is regulated by intracellular cAMP.
Asunto(s)
AMP Cíclico/química , Canales de Potasio Éter-A-Go-Go/química , Canal de Potasio KCNQ1/química , 1-Metil-3-Isobutilxantina/administración & dosificación , Potenciales de Acción/fisiología , Animales , Células CHO , Células Cultivadas , Colforsina/administración & dosificación , Cricetinae , Cricetulus , AMP Cíclico/agonistas , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/fisiología , Femenino , Células HEK293 , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Canal de Potasio KCNQ1/fisiología , Masculino , Inhibidores de Fosfodiesterasa/administración & dosificación , ConejosRESUMEN
We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.
Asunto(s)
Potenciales de Acción/fisiología , Regulación hacia Abajo/fisiología , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/fisiología , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/fisiología , Mutación/genética , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Regulación hacia Abajo/genética , Canal de Potasio ERG1 , Electrofisiología , Femenino , Eliminación de Gen , Humanos , Riñón/citología , Riñón/fisiología , Ovario/citología , Ovario/fisiología , Técnicas de Placa-Clamp , TransfecciónRESUMEN
Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin- and caveolin-dependent mechanisms.