RESUMEN
For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The C: omprehensive I: n Vitro P: roarrhythmia A: ssay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell-derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.
Asunto(s)
Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/diagnóstico , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Corazón/efectos de los fármacos , Animales , Humanos , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/diagnóstico , Torsades de Pointes/inducido químicamente , Torsades de Pointes/diagnósticoRESUMEN
Vanoxerine has been in clinical trials for Parkinsonism, depression and cocaine addiction but lacked efficacy. Although a potent blocker of hERG, it produced no serious adverse events. We attributed the unexpected result to offsetting Multiple Ion Channel Effects (MICE). Vanoxerine's effects were strongly frequency-dependent and we repositioned it for treatment of atrial fibrillation and flutter. Vanoxerine terminated AF/AFL in an animal model and a dose-ranging clinical trial. Reversion to normal rhythm was associated with QT prolongation yet absent proarrhythmia markers for Torsade de Pointes (TdP). To understand the QT/TdP discordance, we used quantitative profiling and compared vanoxerine with dofetilide, a selective hERG-blocking torsadogen used for intractable AF, verapamil, a non-torsadogenic MICE comparator and bepridil, a torsadogenic MICE comparator. At clinically relevant concentrations, verapamil blocked hCav1.2 and hERG, as did vanoxerine and bepridil both of which also blocked hNav1.5. In acute experiments and simulations, dofetilide produced early after depolarizations (EADs) and arrhythmias, whereas verapamil, vanoxerine and bepridil produced no proarrhythmia markers. Of the MICE drugs only bepridil inhibited hERG trafficking following overnight exposure. The results are consistent with the emphasis on MICE of the CiPA assay. Additionally we propose that trafficking inhibition of hERG be added to CiPA.
Asunto(s)
Corazón/efectos de los fármacos , Canales Iónicos/metabolismo , Miocardio/metabolismo , Piperazinas/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Bepridil/farmacología , Células CHO , Simulación por Computador , Cricetulus , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Concentración 50 Inhibidora , Potenciales de la Membrana/efectos de los fármacos , Modelos Biológicos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Técnicas de Placa-Clamp , Fenetilaminas/farmacología , Sulfonamidas/farmacología , Verapamilo/farmacologíaRESUMEN
Cardiac toxicity is a leading contributor to late-stage attrition in the drug discovery process and to withdrawal of approved from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply. But human stem cell-derived cardiomyocytes (SC-hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery and safety pharmacology. As a result, SC-hCM electrophysiology has become a valuable tool to assess cardiac risk associated with drugs. This unit describes techniques for recording individual currents carried by sodium, calcium and potassium ions, as well as single cell action potentials, and impedance recordings from contracting syncytia of thousands of interconnected cells.
Asunto(s)
Miocitos Cardíacos/fisiología , Células Madre/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocitos Cardíacos/efectos de los fármacos , Técnicas de Placa-ClampRESUMEN
Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/fisiología , Modelos Teóricos , Torsades de Pointes/fisiopatología , Canal de Potasio ERG1 , Humanos , Técnicas de Placa-Clamp , Valor Predictivo de las Pruebas , Torsades de Pointes/diagnósticoRESUMEN
Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe²âº on permeation and gating of Ca(V)3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe²âº state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe²âº rapidly blocked currents with 2 mM extracellular Ca²âº in a voltage-dependent manner, as described by a Woodhull model with K(D) = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe²âº also shifted activation to more-depolarized voltages (by â¼10 mV with 1.8 mM extracellular Fe²âº) somewhat more strongly than did extracellular Ca²âº or Mg²âº, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e(-)/98 Ų and K(Fe) = 4.5 M⻹ for extracellular Fe²âº. In the absence of extracellular Ca²âº (and with extracellular Na⺠replaced by TEA), Fe²âº carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at -60 mV with 10 mM extracellular Fe²âº). With a two-site/three-barrier Eyring model for permeation of Ca(V)3.1 channels, we estimated a transport rate for Fe²âº of â¼20 ions/s for each open channel at -60 mV and pH 7.2, with 1 µM extracellular Fe²âº (with 2 mM extracellular Ca²âº). Because Ca(V)3.1 channels exhibit a significant "window current" at that voltage (open probability, â¼1%), Ca(V)3.1 channels represent a likely pathway for Fe²âº entry into cells with clinically relevant concentrations of extracellular Fe²âº.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Compuestos Ferrosos/metabolismo , Compuestos Ferrosos/farmacología , Transferrina/metabolismo , Bario/metabolismo , Línea Celular , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Magnesio/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Técnicas de Placa-Clamp/métodosRESUMEN
We explored the ability of a two-site, three-barrier (2S3B) Eyring model to describe recently reported data on current flow through open Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide range (100 nM: -110 mM: ) while recording whole-cell currents over a wide voltage range (-150 mV to +100 mV) from channels stably expressed in HEK 293 cells. Effects on permeation were isolated using instantaneous current-voltage relationships (IIV) after strong, brief depolarizations to activate channels with minimal inactivation. Most experimental results were reproduced by a 2S3B model. The model described the IIV relationships, apparent affinities for permeation and block for Ca(2+) and Ba(2+), and shifts in reversal potential between Ca(2+) and Ba(2+). The fit to block by 1 mM Mg(2+)(i) was reasonable, but block by Mg(2+)(0) was described less well. Surprisingly, fits were comparable with strong ion-ion repulsion, with no repulsion, or with intermediate values. With weak repulsion, there was a single high-affinity site, with a low-affinity site near the cytoplasmic side of the pore. With strong repulsion, the net charge of ions in the pore was near +2 over a relatively wide range of concentration and voltage, suggesting a knockoff mechanism. With strong repulsion, Ba(2+) preferred the inner site, while Ca(2+) preferred the outer site, potentially explaining faster entry of Ni(2+) and other pore blockers when Ba(2+) is the charge carrier.
Asunto(s)
Bario/metabolismo , Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Magnesio/metabolismo , Modelos Biológicos , Sodio/metabolismo , Línea Celular , HumanosRESUMEN
We examined the concentration dependence of currents through Ca(V)3.1 T-type calcium channels, varying Ca(2+) and Ba(2+) over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current-voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by P(Ca)/P(Na) = 87 and P(Ca)/P(Ba) = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent K(d) values were similar for Ca(2+) and Ba(2+), both for block of currents carried by Na(+) (3 muM for Ca(2+) vs. 4 muM for Ba(2+), at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca(2+) vs. 2.5 mM for Ba(2+); nearly voltage independent). Block by 3-10 muM Ca(2+) was time dependent, described by bimolecular kinetics with binding at approximately 3 x 10(8) M(-1)s(-1) and voltage-dependent exit. Ca(2+)(o), Ba(2+)(o), and Mg(2+)(o) also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e(-) per 98 A(2) from Gouy-Chapman theory. Additionally, inward currents inactivated approximately 35% faster in Ba(2+)(o) (vs. Ca(2+)(o) or Na(+)(o)). The accelerated inactivation in Ba(2+)(o) correlated with the transition from Na(+) to Ba(2+) permeation, suggesting that Ba(2+)(o) speeds inactivation by occupying the pore. We conclude that the selectivity of the "surface charge" among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca(2+) or Ba(2+).
Asunto(s)
Bario/farmacología , Canales de Calcio Tipo T/metabolismo , Calcio/farmacología , Activación del Canal Iónico/efectos de los fármacos , Magnesio/farmacología , Sodio/farmacología , Bario/metabolismo , Calcio/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Magnesio/metabolismo , Potenciales de la Membrana/fisiología , Permeabilidad , Sodio/metabolismoRESUMEN
Ni(2+) inhibits current through calcium channels, in part by blocking the pore, but Ni(2+) may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni(2+) on Ca(V)3.1 is affected by permeant ions. We find two components to block by Ni(2+), a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent K(d) = 3 vs. 1 mM Ni(2+), with 2 mM Ca(2+) or Ba(2+)) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca(2+) or Ba(2+)). Slow block depends both on the concentration and on the identity of the permeant ion (Ca(2+) vs. Ba(2+) vs. Na(+)). Slow block is 2-3x faster in Ba(2+) than in Ca(2+) (2 or 110 mM), and is approximately 10x faster with 2 vs. 110 mM Ca(2+) or Ba(2+). Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations ( approximately 3 muM Ca(2+)). We conclude that both fast and slow block of Ca(V)3.1 by Ni(2+) are most consistent with occlusion of the pore. The exit rate of Ni(2+) for slow block is reduced at high Ni(2+) concentrations, suggesting that the site responsible for fast block can "lock in" slow block by Ni(2+), at a site located deeper within the pore. In contrast to the complex pore block observed for Ca(V)3.1, inhibition of Ca(V)3.2 by Ni(2+) was essentially independent of voltage, and was similar in 2 mM Ca(2+) vs. Ba(2+), consistent with inhibition by a different mechanism, at a site outside the pore.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/metabolismo , Níquel/farmacología , Bario/farmacología , Calcio/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Activación del Canal Iónico , Potenciales de la Membrana/efectos de los fármacosRESUMEN
The cardiac sodium channel Nav 1.5 is essential for the physiological function of the heart and contributes to lethal cardiac arrhythmias and sudden death when mutated. Here, we report that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and modulates the physiological function of Nav 1.5. The yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domains DII and DIII) of Nav 1.5. The interaction was further demonstrated by both in vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation assays in both HEK293 cells with co-expression of MOG1 and Nav1.5 and native cardiac cells. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities. In neonatal myocytes, overexpression of MOG1 increased current densities nearly 2-fold. Western blot analysis revealed that MOG1 increased cell surface expression of Nav1.5, which may be the underlying mechanism by which MOG1 increased sodium current densities. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues with predominant localization at the intercalated discs. In cardiomyocytes, MOG1 is mostly localized in the cell membrane and co-localized with Nav1.5. These results indicate that MOG1 is a critical regulator of sodium channel function in the heart and reveal a new cellular function for MOG1. This study further demonstrates the functional diversity of Nav1.5-binding proteins, which serve important functions for Nav1.5 under different cellular conditions.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Musculares/química , Canales de Sodio/química , Proteína de Unión al GTP ran/fisiología , Animales , Animales Recién Nacidos , Electrofisiología/métodos , Glutatión Transferasa/metabolismo , Corazón/fisiología , Humanos , Ratones , Ratones Endogámicos CBA , Modelos Biológicos , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas del Sistema de Dos Híbridos , Proteína de Unión al GTP ran/químicaRESUMEN
Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular "pore gate" has also been proposed. We have used the voltage dependence of block by extracellular Y(3+) as a tool to locate the activation gate of the alpha1G (Ca(V)3.1) T-type calcium channel. Y(3+) block exhibited no clear voltage dependence from -40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y(3+) at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca(2+) was replaced with Ba(2+), the rate of open block by Y(3+) was unaffected, but closed block was threefold faster than in Ca(2+), suggesting the slower closed-block rate reflects ion-ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Itrio/farmacología , Animales , Canales de Calcio Tipo T/efectos de los fármacos , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Líquido Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Riñón , Potenciales de la Membrana/efectos de los fármacos , RatasRESUMEN
Apoptosis results in cell shrinkage and intracellular acidification, processes opposed by the ubiquitously expressed NHE1 Na(+)/H(+) exchanger. In addition to mediating Na(+)/H(+) transport, NHE1 interacts with ezrin/radixin/moesin (ERM), which tethers NHE1 to cortical actin cytoskeleton to regulate cell shape, adhesion, motility, and resistance to apoptosis. We hypothesize that apoptotic stress activates NHE1-dependent Na(+)/H(+) exchange, and NHE1-ERM interaction is required for cell survival signaling. Apoptotic stimuli induced NHE1-regulated Na(+)/H(+) transport, as demonstrated by ethyl-N-isopropyl-amiloride-inhibitable, intracellular alkalinization. Ectopic NHE1, but not NHE3, expression rescued NHE1-null cells from apoptosis induced by staurosporine or N-ethylmaleimide-stimulated KCl efflux. When cells were subjected to apoptotic stress, NHE1 and phosphorylated ERM physically associated within the cytoskeleton-enriched fraction, resulting in activation of the pro-survival kinase, Akt. NHE1-associated Akt activity and cell survival were inhibited in cells expressing ERM binding-deficient NHE1, dominant negative ezrin constructs, or ezrin mutants with defective binding to phosphoinositide 3-kinase, an upstream regulator of Akt. We conclude that NHE1 promotes cell survival by dual mechanisms: by defending cell volume and pH(i) through Na(+)/H(+) exchange and by functioning as a scaffold for recruitment of a signalplex that includes ERM, phosphoinositide 3-kinase, and Akt.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Intercambiadores de Sodio-Hidrógeno/fisiología , Apoptosis , Adhesión Celular , Línea Celular , Supervivencia Celular , Citoesqueleto/metabolismo , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Etilmaleimida/farmacología , Humanos , Concentración de Iones de Hidrógeno , Immunoblotting , Modelos Biológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Plásmidos/metabolismo , Cloruro de Potasio/farmacología , Pruebas de Precipitina , Unión Proteica , Proteínas Proto-Oncogénicas c-akt , Interferencia de ARN , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
To evaluate the effects of the iron chelator deferoxamine on the functional and structural manifestations of iron-induced cardiac dysfunction, we measured cardiac power, left ventricular systolic, and diastolic function as (dP/dt)max and (dP/dt)min, respectively, and left ventricular and septal wall thickness in isolated heart preparations derived from the Mongolian gerbil model of iron overload. We induced iron overload with weekly subcutaneous injections of iron dextran (800 mg/kg/wk); deferoxamine (DFO; 100 mg/kg) was administered twice daily by subcutaneous injection, 5 of 7 days each week; and control animals received weekly subcutaneous injections of dextran alone. Animals administered iron alone initially exhibited, at 5 weeks, increased cardiac power but by 12 to 20 weeks, cardiac power was severely diminished, with impairment of both systolic and diastolic function of the left ventricle and marked cardiac hypertrophy (P<.001 for all vs control animals). Administration of DFO with iron did not interfere with the initial augmentation of cardiac power at 5 weeks but prevented the subsequent deterioration in cardiac performance. After 12 to 20 weeks, gerbils given DFO with iron had mean values of cardiac power indistinguishable from those of control animals; both systolic and diastolic function were significantly enhanced not only in comparison with those of animals treated with iron alone but also with respect to controls. In addition, DFO prevented cardiac hypertrophy; mean ventricular and septal wall thickness in gerbils given DFO and iron were not significantly different from those in controls. In the gerbil model of iron overload, concurrent administration of DFO with iron prevents both the development of cardiac hypertrophy and the progressive deterioration in cardiac performance that are produced by chronic iron accumulation.
Asunto(s)
Cardiomegalia/tratamiento farmacológico , Cardiomegalia/prevención & control , Deferoxamina/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/prevención & control , Quelantes del Hierro/farmacología , Animales , Cardiomegalia/patología , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/patología , Cardiomiopatías/prevención & control , Femenino , Gerbillinae , Insuficiencia Cardíaca/patología , Hierro/farmacología , Sobrecarga de Hierro/complicaciones , Contracción Miocárdica/efectos de los fármacosRESUMEN
We investigated the time course of electrocardiographic (ECG) changes in the Mongolian gerbil model of iron overload and the effects of the iron chelator deferoxamine (DFO) on these changes. Iron overload was produced with weekly subcutaneous injections of low doses (200 mg/kg/wk) or high doses (800 mg/kg/wk) of iron-dextran. DFO was administered subcutaneously at a dose of 200 mg/kg/day to high-dose animals. Our results show that (1) survival of iron-overloaded gerbils is dose-dependent, with median survival times of 68 and 14 weeks for low- and high-dose animals, respectively; (2) both low and high doses produce prolongation of the PR interval and bradycardia in early stages and prolongation of the QT interval, premature ventricular contractions, variable degrees of atrioventricular block, changes in the ST segment, and T-wave inversion at later stages coinciding with the development of heart failure; (3) DFO prevented death during 20 weeks of high-dose iron-dextran; (4) DFO prevented ECG changes, although delayed prolongation of PR intervals and QRS complexes occurred; and (5) despite marked prolongation of survival and prevention of ECG changes, DFO had modest effects on total cardiac iron content. We speculate that DFO chelates a small iron pool located within the cytoplasm of iron-overloaded cardiomyocytes.
Asunto(s)
Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/etiología , Deferoxamina/uso terapéutico , Electrocardiografía , Quelantes del Hierro/uso terapéutico , Sobrecarga de Hierro/complicaciones , Animales , Cardiomiopatías/fisiopatología , Deferoxamina/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Gerbillinae , Frecuencia Cardíaca , Hierro/análisis , Quelantes del Hierro/administración & dosificación , Miocardio/química , Tasa de SupervivenciaRESUMEN
Mutations in the human ether-a-gogo-related gene (HERG) K(+) channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduce I(Kr) currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K(+) channels without block.