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1.
FASEB J ; 33(2): 2537-2552, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30289750

RESUMEN

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (KV) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout ( Kcne5-/0) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5+/0 mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte KV current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current ( Ito,f). The current increases arose from an apex-specific increase in slow transient outward current-1 ( IKslow,1) (conducted by KV1.5) and Ito,f (conducted by KV4) and an increase in IKslow,2 (conducted by KV2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where KV2.1 was also detected in both Kcne5-/0 and Kcne5+/0 mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and KV2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases IK,slow2 by reducing KV2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of KV2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in KV2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of KV2.1 in mouse heart. Increased KV current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS.-David, J.-P., Lisewski, U., Crump, S. M., Jepps, T. A., Bocksteins, E., Wilck, N., Lossie, J., Roepke, T. K., Schmitt, N., Abbott, G. W. Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular KV currents and predisposes to ventricular arrhythmia.


Asunto(s)
Fibrilación Atrial/complicaciones , Síndrome de Brugada/complicaciones , Genes Ligados a X , Activación del Canal Iónico , Miocitos Cardíacos/patología , Canales de Potasio con Entrada de Voltaje/fisiología , Taquicardia Ventricular/etiología , Animales , Fibrilación Atrial/genética , Síndrome de Brugada/genética , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Potasio/metabolismo , Eliminación de Secuencia , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patología
2.
Pacing Clin Electrophysiol ; 43(2): 210-216, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31899541

RESUMEN

BACKGROUND: The slow delayed rectifier potassium current IKs is crucial for the repolarization of the cardiac action potential. It is conducted by the voltage-gated channel KV 7.1 encoded by KCNQ1, together with its ß-subunit KCNE1. Loss-of-function (LOF) mutations in KCNQ1 have been associated with heritable cardiac arrhythmias such as Long QT syndrome (LQTS). This disease is characterized by prolonged ventricular repolarization and propensity to ventricular tachyarrhythmia that may lead to syncope, cardiac arrest, and sudden death. We aimed to functionally characterize two KV 7.1 mutations (p.A150T and p.L374H) identified in two independent LQTS patients with different severity of disease phenotype, family history, and co-segregation of LQTS. METHODS: We performed whole-cell patch clamp recordings in CHO-K1 cells, and confocal imaging in Madin-Darby Canine Kidney (MDCK) cells. RESULTS: IKs -A150T showed significantly decreased current amplitudes from above +20 mV (approximately 52% decrease at +40 mV), but demonstrated cell membrane localization similar to wild-type (WT). IKs -L374H, however, exhibited a complete LOF compared to WT channels. Confocal imaging showed endoplasmic reticulum retention of the channel in MDCK cells. Mimicking the heterozygous state of the patients by co-expressing WT and mutant subunits resulted in an approximately 22% decrease in current at +40 mV for A150T. The L374H mutation showed a more pronounced effect (62% reduction at +40 mV compared to WT channel). CONCLUSION: Both mutations, KV 7.1 A150T and L374H, led to loss of channel function. The degree of LOF may mirror the disease phenotype observed in the patients.


Asunto(s)
Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/genética , Canales de Potasio con Entrada de Voltaje/genética , Potenciales de Acción , Adulto , Animales , Células CHO , Cricetulus , Perros , Humanos , Mutación con Pérdida de Función , Células de Riñón Canino Madin Darby , Persona de Mediana Edad , Técnicas de Placa-Clamp , Linaje , Fenotipo
6.
Traffic ; 14(4): 399-411, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23324056

RESUMEN

The voltage-gated potassium channel KV 7.1 is regulated by non-pore forming regulatory KCNE ß-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current IKs . However, where the subunits assemble and which of the subunits determines localization of the IKs -complex has not been unequivocally resolved yet. We employed trafficking-deficient KV 7.1 and KCNE1 mutants to investigate IKs trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post-endoplasmic reticulum compartment. We demonstrate that KV 7.1 targets the IKs -complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical KV 7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that KV 7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of KV 7.1/KCNE complexes.


Asunto(s)
Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Animales , Membrana Celular/metabolismo , Perros , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/genética , Células de Riñón Canino Madin Darby , Mutación Missense , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Dominios y Motivos de Interacción de Proteínas , Señales de Clasificación de Proteína/genética , Subunidades de Proteína/genética , Transporte de Proteínas , Vías Secretoras
7.
Am J Physiol Cell Physiol ; 303(9): C963-76, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-22914645

RESUMEN

The roles of several small GTPases in the expression of an endogenous potassium current, I(to,f), in adult rat ventricular myocytes have been investigated. The results indicate that forward trafficking of newly synthesized Kv4.2, which underlies I(to,f) in these cells, requires both Rab1 and Sar1 function. Expression of a Rab1 dominant negative (DN) reduced I(to,f) current density by roughly one-half relative to control, mCherry-transfected myocytes. Similarly, expression of a Sar1DN nearly halved I(to,f) current density. Rab11 is not essential to trafficking of Kv4.2, as expression of a Rab11DN had no effect on I(to,f) over the time frames investigated here. In a process dependent on intact endoplasmic reticulum (ER)-to-Golgi transport, however, overexpression of wild-type Rab11 resulted in a doubling of I(to,f) density; block of ER-to-Golgi traffic by Brefeldin A completely abrogated the effect. Also implicated in the trafficking of Kv4.2 are Rab5 and Rab4. Rab5DN expression increased endogenous I(to,f) by two- to threefold, nonadditively with inhibition of dynamin-dependent endocytosis. And, in a phenomenon similar to that previously reported for myoblast-expressed Kv1.5, Rab4DN expression roughly doubled endogenous peak transient currents. Colocalization experiments confirmed the involvement of Rab4 in postinternalization trafficking of Kv4.2. There was little role evident for the lysosome in the degradation of internalized Kv4.2, as overexpression of neither wild-type nor DN isoforms of Rab7 had any effect on I(to,f). Instead, degradation may depend largely on the proteasome; the proteasome inhibitor MG132 significantly increased I(to,f) density.


Asunto(s)
Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Transporte de Proteínas/fisiología , Canales de Potasio Shal/metabolismo , Animales , Brefeldino A/farmacología , Células Cultivadas , Retículo Endoplásmico/metabolismo , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/metabolismo , Ventrículos Cardíacos/efectos de los fármacos , Leupeptinas/farmacología , Masculino , Proteínas de Unión al GTP Monoméricas/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Proteínas de Unión al GTP rab5/metabolismo
8.
BMC Med Genet ; 13: 24, 2012 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-22471742

RESUMEN

BACKGROUND: Atrial fibrillation (AF) is the most common arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutations in KV7.1, the pore-forming α-subunit of the IKs channel, have been associated with AF. We hypothesized that early-onset lone AF is associated with mutations in the IKs channel regulatory subunit KCNE1. METHODS: In 209 unrelated early-onset lone AF patients (< 40 years) the entire coding sequence of KCNE1 was bidirectionally sequenced. We analyzed the identified KCNE1 mutants electrophysiologically in heterologous expression systems. RESULTS: Two non-synonymous mutations G25V and G60D were found in KCNE1 that were not present in the control group (n = 432 alleles) and that have not previously been reported in any publicly available databases or in the exom variant server holding exom data from more than 10.000 alleles. Proband 1 (female, age 45, G25V) had onset of paroxysmal AF at the age of 39 years. Proband 2 (G60D) was diagnosed with lone AF at the age of 33 years. The patient has inherited the mutation from his mother, who also has AF. Both probands had no mutations in genes previously associated with AF. In heterologous expression systems, both mutants showed significant gain-of-function for IKs both with respect to steady-state current levels, kinetic parameters, and heart rate-dependent modulation. CONCLUSIONS: Mutations in KV7.1 leading to gain-of-function of IKs current have previously been described in lone AF, yet this is the first time a mutation in the beta-subunit KCNE1 is associated with the disease. This finding further supports the hypothesis that increased potassium current enhances AF susceptibility.


Asunto(s)
Fibrilación Atrial/epidemiología , Fibrilación Atrial/genética , Predisposición Genética a la Enfermedad , Mutación/genética , Canales de Potasio con Entrada de Voltaje/genética , Subunidades de Proteína/genética , Adulto , Edad de Inicio , Fibrilación Atrial/fisiopatología , Secuencia de Bases , Estudios de Cohortes , Análisis Mutacional de ADN , Dinamarca/epidemiología , Conductividad Eléctrica , Femenino , Humanos , Activación del Canal Iónico , Cinética , Masculino , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Linaje
9.
Can J Physiol Pharmacol ; 89(10): 723-36, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21895525

RESUMEN

BACKGROUND: Mutations in the SCN5A gene have been linked to Brugada syndrome (BrS), conduction disease, Long QT syndrome (LQT3), atrial fibrillation (AF), and to pre- and neonatal ventricular arrhythmias. OBJECTIVE: The objective of this study is to characterize a novel mutation in Na(v)1.5 found in a newborn with fetal chaotic atrial tachycardia, post-partum intraventricular conduction delay, and QT interval prolongation. METHODS: Genomic DNA was isolated and all exons and intron borders of 15 ion-channel genes were sequenced, revealing a novel missense mutation (Q270K) in SCN5A. Na(v)1.5 wild type (WT) and Q270K were expressed in CHO-K1 with and without the Na(v)ß1 subunit. Results. Patch-clamp analysis showed ∼40% reduction in peak sodium channel current (I(Na)) density for Q270K compared with WT. Fast and slow decay of I(Na) were significantly slower in Q270K. Steady-state activation and inactivation of Q270K channels were shifted to positive potentials, and window current was increased. The tetrodotoxin-sensitive late I(Na) was increased almost 3-fold compared with WT channels. Ranolazine reduced late I(Na) in WT and Q270K channels, while exerting minimal effects on peak I(Na). CONCLUSION: The Q270K mutation in SCN5A reduces peak I(Na) while augmenting late I(Na), and may thus underlie the development of atrial tachycardia, intraventricular conduction delay, and QT interval prolongation in an infant.


Asunto(s)
Arritmias Cardíacas/genética , Potenciales de la Membrana/genética , Mutación Missense/genética , Canales de Sodio/genética , Animales , Células CHO , Línea Celular Transformada , Cricetinae , Análisis Mutacional de ADN , Femenino , Humanos , Recién Nacido , Canales Iónicos/genética , Potenciales de la Membrana/fisiología , Canal de Sodio Activado por Voltaje NAV1.5 , Técnicas de Placa-Clamp/métodos , Síndrome
10.
Sci Rep ; 5: 12813, 2015 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-26242757

RESUMEN

The diversity of the voltage-gated K(+) (Kv) channel subfamily Kv2 is increased by interactions with auxiliary ß-subunits and by assembly with members of the modulatory so-called silent Kv subfamilies (Kv5-Kv6 and Kv8-Kv9). However, it has not yet been investigated whether these two types of modulating subunits can associate within and modify a single channel complex simultaneously. Here, we demonstrate that the transmembrane ß-subunit KCNE5 modifies the Kv2.1/Kv6.4 current extensively, whereas KCNE2 and KCNE4 only exert minor effects. Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation. In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers. Co-localization of Kv2.1, Kv6.4 and KCNE5 was demonstrated with immunocytochemistry and formation of Kv2.1/Kv6.4/KCNE5 and Kv2.1/KCNE5 complexes was confirmed by Fluorescence Resonance Energy Transfer experiments performed in HEK293 cells. These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed. In vivo, formation of such tripartite Kv2.1/Kv6.4/KCNE5 channel complexes might contribute to tissue-specific fine-tuning of excitability.


Asunto(s)
Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio Shab/metabolismo , Animales , Células HEK293 , Humanos , Potenciales de la Membrana , Ratones , Canales de Potasio con Entrada de Voltaje/fisiología , Estructura Cuaternaria de Proteína , Subunidades de Proteína/fisiología , Transporte de Proteínas
11.
Sci Rep ; 5: 10009, 2015 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-26066609

RESUMEN

The Long QT syndrome (LQTS) is a disorder characterized by a prolongation of the QT interval and a propensity to ventricular tachyarrhythmias, which may lead to syncope, cardiac arrest, or sudden death. Our objective was to (1) determine the incidence of variants with unknown significance (VUS) in a cohort of consecutive LQTS patients and (2) to determine the percentage of those with novel missense VUS that have demonstrable functional channel abnormalities from a single referral center. We performed genetic screening of candidate genes in 39 probands with a diagnosis of LQTS to identify mutations and variants. Seven variants of unknown significance were identified, six were missense variants and one was a splice site variant. We investigated the six novel missense VUS in five patients; three missense variants in KCNQ1 (L236R, W379R, Y522S) and three missense variants in KCNH2 (R35W, S620G, V491I). We employed two-electrode voltage-clamp experiments in Xenopus laevis oocytes and confocal imaging to characterize the novel missense mutations functionally. We revealed electrophysiological and trafficking loss-of-function phenotypes. This report emphasizes the frequency of adverse channel function in patients with LQTS and the importance of heterologous studies to define channel function.


Asunto(s)
Canales de Potasio Éter-A-Go-Go/genética , Canal de Potasio KCNQ1/genética , Síndrome de QT Prolongado/genética , Mutación Missense , Sustitución de Aminoácidos , Animales , Estudios de Cohortes , Canal de Potasio ERG1 , Femenino , Humanos , Masculino , Xenopus laevis
12.
Biomark Med ; 8(4): 557-70, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24796621

RESUMEN

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Disturbances in cardiac potassium conductance are considered as one of the disease mechanisms in AF. We aimed to investigate if mutations in potassium-channel ß-subunits KCNE2 and KCNE3 are associated with early-onset lone AF. METHODS & RESULTS: The coding regions of KCNE2 and KCNE3 were bidirectionally sequenced in 192 unrelated patients diagnosed with early-onset lone AF (<40 years). Two nonsynonymous missense mutations were identified in KCNE2 (M23L and I57T). Both mutations were absent in a healthy control group (n=1500 alleles). Electrophysiological investigations were performed for both mutations in combination with candidate pore-forming α-subunits KV7.1, KV11.1, KV4.3 and KV1.5. A significant gain-of-function effect was observed upon coexpression with KV7.1 and KV7.1+KCNE1. Confocal imaging found no differences in subcellular localization. No disease-suspected mutations were identified in KCNE3. CONCLUSION: We identified two KCNE2 gain-of-function missense mutations that seem to increase the susceptibility of early-onset lone AF. These results confirm previous findings indicating that gain-of-function in the slow delayed rectifier potassium current might be involved in the pathogenesis of AF.


Asunto(s)
Fibrilación Atrial/diagnóstico , Fibrilación Atrial/genética , Canales de Potasio con Entrada de Voltaje/genética , Adulto , Alelos , Secuencia de Aminoácidos , Animales , Fibrilación Atrial/metabolismo , Secuencia de Bases , Presión Sanguínea , Células CHO , Cricetinae , Cricetulus , Análisis Mutacional de ADN , Femenino , Predisposición Genética a la Enfermedad , Humanos , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ1/metabolismo , Masculino , Microscopía Confocal , Mutación Missense , Canales de Potasio con Entrada de Voltaje/metabolismo
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