RESUMO
The KCNQ1 gene encodes a voltage-gated potassium channel required for cardiac action potentials. Mutations in this gene have been associated with hereditary long QT syndrome 1, Jervell and Lange-Nielsen syndromes, and familial atrial fibrillation. The NM_000218.3(KCNQ1): c.604 + 2T > C mutation has been categorized as the causative variant leading to LQT1. In this study, we generated a KCNQ1 (c.644 + 2T > C) mutation human embryonic stem cell line WAe009-A-1L based on CRISPR base editing system. WAe009-A-1L cell has the potential to differentiate cardiomyocytes and would be used as an in vitro disease model for mechanism exploration and drug screening.
Assuntos
Edição de Genes , Células-Tronco Embrionárias Humanas , Canal de Potássio KCNQ1 , Mutação , Humanos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Edição de Genes/métodos , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Linhagem Celular , Sistemas CRISPR-Cas , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Diferenciação Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genéticaRESUMO
Voltage-gated K+ (KV) channels govern K+ ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our findings demonstrate that KV cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types.
Assuntos
Subunidades Proteicas , Humanos , Animais , Subunidades Proteicas/metabolismo , Células HEK293 , Potenciais da Membrana , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canal de Potássio KCNQ1/metabolismo , Canal de Potássio KCNQ1/genéticaRESUMO
Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1-/- mice displayed altered action potential firing properties. Strikingly, only male KCNQ1-/- mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8-/- animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.
Assuntos
Temperatura Baixa , Canal de Potássio KCNQ1 , Animais , Masculino , Feminino , Camundongos , Canal de Potássio KCNQ1/metabolismo , Canal de Potássio KCNQ1/genética , Camundongos Knockout , Gânglios Espinais/metabolismo , Sensação Térmica/fisiologia , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPM/genética , Camundongos Endogâmicos C57BL , Potenciais de Ação/fisiologia , Caracteres Sexuais , Mentol/farmacologiaRESUMO
The use of tyrosine kinase inhibitors (TKIs) has resulted in significant occurrence of arrhythmias. However, the precise mechanism of the proarrhythmic effect is not fully understood. In this study, we found that nilotinib (NIL), vandetanib (VAN), and mobocertinib (MOB) induced the development of "cellrhythmia" (arrhythmia-like events) in a concentration-dependent manner in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Continuous administration of NIL, VAN, or MOB in animals significantly prolonged the action potential durations (APD) and increased susceptibility to arrhythmias. Using phosphoproteomic analysis, we identified proteins with altered phosphorylation levels after treatment with 3⯵M NIL, VAN, and MOB for 1.5â¯h. Using these identified proteins as substrates, we performed kinase-substrate enrichment analysis to identify the kinases driving the changes in phosphorylation levels of these proteins. MAPK and WNK were both inhibited by NIL, VAN, and MOB. A selective inhibitor of WNK1, WNK-IN-11, induced concentration- and time-dependent cellrhythmias and prolonged field potential duration (FPD) in hiPSC-CMs in vitro; furthermore, administration in guinea pigs confirmed that WNK-IN-11 prolonged ventricular repolarization and increased susceptibility to arrhythmias. Fingding indicated that WNK1 inhibition had an in vivo and in vitro arrhythmogenic phenotype similar to TKIs. Additionally,three of TKIs reduced hERG and KCNQ1 expression at protein level, not at transcription level. Similarly, the knockdown of WNK1 decreased hERG and KCNQ1 protein expression in hiPSC-CMs. Collectively, our data suggest that the proarrhythmic effects of NIL, VAN, and MOB occur through a kinase inhibition mechanism. NIL, VAN, and MOB inhibit WNK1 kinase, leading to a decrease in hERG and KCNQ1 protein expression, thereby prolonging action potential repolarization and consequently cause arrhythmias.
Assuntos
Potenciais de Ação , Arritmias Cardíacas , Miócitos Cardíacos , Piperidinas , Proteômica , Pirimidinas , Quinazolinas , Humanos , Arritmias Cardíacas/induzido quimicamente , Animais , Proteômica/métodos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Piperidinas/farmacologia , Piperidinas/toxicidade , Pirimidinas/toxicidade , Pirimidinas/farmacologia , Quinazolinas/toxicidade , Quinazolinas/farmacologia , Potenciais de Ação/efeitos dos fármacos , Inibidores de Proteínas Quinases/toxicidade , Inibidores de Proteínas Quinases/farmacologia , Fosforilação , Canal de Potássio ERG1/metabolismo , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/genética , Cobaias , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Canal de Potássio KCNQ1/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/efeitos dos fármacos , Fosfoproteínas/metabolismo , Relação Dose-Resposta a DrogaRESUMO
Long QT Syndrome (LQTS) is a genetic heart disorder that can induce cardiac arrhythmias. The most prevalent subtype, LQT1, stems from rare variants in the KCNQ1 gene. Utilizing induced pluripotent stem cells (iPSCs) enables detailed cellular studies and personalized medicine approaches for this life-threatening condition. We generated two LQT1 iPSC lines with single nucleotide nonsense mutations, c.1031 C > T and c.1121 T > A in KCNQ1. Both lines exhibited typical iPSC morphology, expressed high levels of pluripotent markers, maintained normal karyotype, and possessed the capability to differentiate into three germ layers. These cell lines serve as important tools for investigating the biological mechanisms underlying LQT1 due to mutations in the KCNQ1 gene.
Assuntos
Células-Tronco Pluripotentes Induzidas , Canal de Potássio KCNQ1 , Síndrome do QT Longo , Humanos , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/genética , Síndrome do QT Longo/patologia , Síndrome do QT Longo/metabolismo , Linhagem Celular , Heterozigoto , Mutação , Masculino , Feminino , Diferenciação CelularAssuntos
Canal de Potássio ERG1 , Canal de Potássio KCNQ1 , Fenótipo , Humanos , Canal de Potássio ERG1/metabolismo , Canal de Potássio ERG1/genética , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Síndrome de Romano-Ward/genética , Síndrome de Romano-Ward/fisiopatologia , Síndrome de Romano-Ward/metabolismo , Síndrome de Romano-Ward/diagnóstico , Predisposição Genética para Doença , Potenciais de Ação , Animais , Mutação , Frequência CardíacaRESUMO
Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.
Assuntos
Proteínas de Ancoragem à Quinase A , Proteínas do Citoesqueleto , Canal de Potássio KCNQ1 , Síndrome do QT Longo , Animais , Feminino , Humanos , Masculino , Proteínas de Ancoragem à Quinase A/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Proteínas de Ancoragem à Quinase A/química , Células CHO , Cricetulus , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Canal de Potássio KCNQ1/química , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Modelos Moleculares , Mutação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ligação ProteicaRESUMO
The KCNQ1 gene encodes a voltage-gated potassium channel, which plays an important role in the repolarization of myocardial action potentials. Mutations in this gene often result in type 1 long QT syndrome (LQT1). Here, we generated a KCNQ1 (c.1032 + 2 T > C) mutant human embryonic stem cell line (WAe009-A-1D) based on the transient expression adenine base editing system that converts base A to G. The WAe009-A-1D cell maintains the morphology, pluripotency, and normal karyotype of the stem cells and is capable of differentiating into all three germ layers in vivo.
Assuntos
Edição de Genes , Células-Tronco Embrionárias Humanas , Canal de Potássio KCNQ1 , Humanos , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Linhagem Celular , Sistemas CRISPR-Cas , Diferenciação Celular , MutaçãoRESUMO
The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.
Assuntos
Rim , Vasodilatação , Animais , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Masculino , Ratos , Camundongos , Rim/metabolismo , Rim/irrigação sanguínea , Canal de Potássio KCNQ1/metabolismo , Isoproterenol/farmacologia , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Camundongos Knockout , Receptores Adrenérgicos beta/metabolismo , Circulação Renal/efeitos dos fármacos , Circulação Renal/fisiologia , Camundongos Endogâmicos C57BL , Ratos Wistar , Hipertensão/fisiopatologia , Hipertensão/metabolismoRESUMO
AIMS: While variants in KCNQ1 are the commonest cause of the congenital long QT syndrome, we and others find only a small IKs in cardiomyocytes from human-induced pluripotent stem cells (iPSC-CMs) or human ventricular myocytes. METHODS AND RESULTS: We studied population control iPSC-CMs and iPSC-CMs from a patient with Jervell and Lange-Nielsen (JLN) syndrome due to compound heterozygous loss-of-function (LOF) KCNQ1 variants. We compared the effects of pharmacologic IKs block to those of genetic KCNQ1 ablation, using JLN cells, cells homozygous for the KCNQ1 LOF allele G643S, or siRNAs reducing KCNQ1 expression. We also studied the effects of two blockers of IKr, the other major cardiac repolarizing current, in the setting of pharmacologic or genetic ablation of KCNQ1: moxifloxacin, associated with a very low risk of drug-induced long QT, and dofetilide, a high-risk drug. In control cells, a small IKs was readily recorded but the pharmacologic IKs block produced no change in action potential duration at 90% repolarization (APD90). In contrast, in cells with genetic ablation of KCNQ1 (JLN), baseline APD90 was markedly prolonged compared with control cells (469 ± 20 vs. 310 ± 16â ms). JLN cells displayed increased sensitivity to acute IKr block: the concentration (µM) of moxifloxacin required to prolong APD90 100 msec was 237.4 [median, interquartile range (IQR) 100.6-391.6, n = 7] in population cells vs. 23.7 (17.3-28.7, n = 11) in JLN cells. In control cells, chronic moxifloxacin exposure (300â µM) mildly prolonged APD90 (10%) and increased IKs, while chronic exposure to dofetilide (5â nM) produced greater prolongation (67%) and no increase in IKs. However, in the siRNA-treated cells, moxifloxacin did not increase IKs and markedly prolonged APD90. CONCLUSION: Our data strongly suggest that KCNQ1 expression modulates baseline cardiac repolarization, and the response to IKr block, through mechanisms beyond simply generating IKs.
Assuntos
Potenciais de Ação , Células-Tronco Pluripotentes Induzidas , Síndrome de Jervell-Lange Nielsen , Canal de Potássio KCNQ1 , Moxifloxacina , Miócitos Cardíacos , Fenetilaminas , Sulfonamidas , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Potenciais de Ação/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Moxifloxacina/farmacologia , Fenetilaminas/farmacologia , Sulfonamidas/farmacologia , Síndrome de Jervell-Lange Nielsen/genética , Síndrome de Jervell-Lange Nielsen/metabolismo , Síndrome de Jervell-Lange Nielsen/fisiopatologia , Bloqueadores dos Canais de Potássio/farmacologia , Fluoroquinolonas/farmacologiaRESUMO
Gain-of-function mutations in the KCNQ1 gene can cause atrial fibrillation. In this study, we generated an induced stem cell line (GRCHJUi001) from one member of an atrial fibrillation family line, whom had heterozygous mutation in the KCNQ1 gene c.625 T > C (p.Ser209Pro), and the cell line showed maintenance of stem cells characterized by morphology, normal karyotype, and pluripotency.
Assuntos
Fibrilação Atrial , Células-Tronco Pluripotentes Induzidas , Humanos , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Linhagem CelularRESUMO
Altered epigenetic mechanisms have been previously reported in growth restricted offspring whose mothers experienced environmental insults during pregnancy in both human and rodent studies. We previously reported changes in the expression of the DNA methyltransferase Dnmt3a and the imprinted genes Cdkn1c (Cyclin-dependent kinase inhibitor 1C) and Kcnq1 (Potassium voltage-gated channel subfamily Q member 1) in the kidney tissue of growth restricted rats whose mothers had uteroplacental insufficiency induced on day 18 of gestation, at both embryonic day 20 (E20) and postnatal day 1 (PN1). To determine the mechanisms responsible for changes in the expression of these imprinted genes, we investigated DNA methylation of KvDMR1, an imprinting control region (ICR) that includes the promoter of the antisense long non-coding RNA Kcnq1ot1 (Kcnq1 opposite strand/antisense transcript 1). Kcnq1ot1 expression decreased by 51% in growth restricted offspring compared to sham at PN1. Interestingly, there was a negative correlation between Kcnq1ot1 and Kcnq1 in the E20 growth restricted group (Spearman's ρ = 0.014). No correlation was observed between Kcnq1ot1 and Cdkn1c expression in either group at any time point. Additionally, there was a 11.25% decrease in the methylation level at one CpG site within KvDMR1 ICR. This study, together with others in the literature, supports that long non-coding RNAs may mediate changes seen in tissues of growth restricted offspring.
Assuntos
Metilação de DNA , RNA Longo não Codificante , Gravidez , Feminino , Humanos , Animais , Ratos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Impressão Genômica , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Rim/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismoRESUMO
The KCNQ1 channel is important for the repolarization phase of the cardiac action potential. Loss of function mutations in KCNQ1 can cause long QT syndrome (LQTS), which can lead to cardiac arrythmia and even sudden cardiac death. We have previously shown that polyunsaturated fatty acids (PUFAs) and PUFA analogs can activate the cardiac KCNQ1 channel, making them potential therapeutics for the treatment of LQTS. PUFAs bind to KCNQ1 at two different binding sites: one at the voltage sensor (Site I) and one at the pore (Site II). PUFA interaction at Site I shifts the voltage dependence of the channel to the left, while interaction at Site II increases maximal conductance. The PUFA analogs, linoleic-glycine and linoleic-tyrosine, are more effective than linoleic acid at Site I, but less effective at Site II. Using both simulations and experiments, we find that the larger head groups of linoleic-glycine and linoleic-tyrosine interact with more residues than the smaller linoleic acid at Site I. We propose that this will stabilize the negatively charged PUFA head group in a position to better interact electrostatically with the positively charges in the voltage sensor. In contrast, the larger head groups of linoleic-glycine and linoleic-tyrosine compared with linoleic acid prevent a close fit of these PUFA analogs in Site II, which is more confined. In addition, we identify several KCNQ1 residues as critical PUFA-analog binding residues, thereby providing molecular models of specific interactions between PUFA analogs and KCNQ1. These interactions will aid in future drug development based on PUFA-KCNQ1 channel interactions.
Assuntos
Síndrome do QT Longo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Humanos , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Coração , Ácidos Graxos Insaturados/metabolismo , Síndrome do QT Longo/genética , Mutação , Ácidos Linoleicos/farmacologiaRESUMO
We compared the expression of six genes in stomach tissue samples between healthy men and women in different age groups to study sexually dimorphic gene expression. Real-Time RT-PCR was used to compare gene expression between men and women. Our results showed that the expression of KCNQ1 (p = 0.01) was significantly higher in non-menopausal women compared to post-menopausal women. In addition, the expression level of the ATP4A gene in men under 35 years was significantly higher than in men above 50 (p = 0.026). Sexually and age dimorphic gene expression in some genes throughout life may affect gastric function.
Assuntos
Mucosa Gástrica , Canal de Potássio KCNQ1 , Masculino , Humanos , Feminino , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Mucosa Gástrica/metabolismo , Estômago , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismoRESUMO
Voltage-gated potassium (KV) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. KV channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore KV channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of KV channel activators with potential applications in the treatment of arrhythmogenic disorders such as long QT syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac IKs channel - a tetrameric potassium channel complex formed by KV7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac IKs channel, and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the IKs channel by generating modified aromatic head groups designed to probe cation-pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the IKs channel through cation-pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.
Assuntos
Síndrome do QT Longo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Humanos , Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Ácidos Graxos Insaturados/metabolismo , Síndrome do QT Longo/genética , Arritmias Cardíacas , TirosinaRESUMO
The voltage-gated potassium channel KvLQT1 encoded by KCNQ1 plays an important role in the repolarization of myocardial action potentials. KCNQ1 mutations can cause Long QT syndrome type 1 (LQT1), which is considered to be the most common causative gene of LQT. In this study, we established a human embryonic stem cell line KCNQ1L114P/+ (WAe009-A-79) carrying a LQT1 related mutation in KCNQ1. The WAe009-A-79 line maintains the morphology, pluripotency, and normal karyotype of stem cells, and can differentiate into all three germ layers in vivo.
Assuntos
Células-Tronco Embrionárias Humanas , Síndrome do QT Longo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Síndrome de Romano-Ward , Humanos , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Síndrome do QT Longo/genética , Síndrome de Romano-Ward/genética , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio KCNQ/genéticaRESUMO
Voltage-dependent ion channels underlie the propagation of action potentials and other forms of electrical activity in cells. In these proteins, voltage sensor domains (VSDs) regulate opening and closing of the pore through the displacement of their positive-charged S4 helix in response to the membrane voltage. The movement of S4 at hyperpolarizing membrane voltages in some channels is thought to directly clamp the pore shut through the S4-S5 linker helix. The KCNQ1 channel (also known as Kv7.1), which is important for heart rhythm, is regulated not only by membrane voltage but also by the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2). KCNQ1 requires PIP2 to open and to couple the movement of S4 in the VSD to the pore. To understand the mechanism of this voltage regulation, we use cryogenic electron microscopy to visualize the movement of S4 in the human KCNQ1 channel in lipid membrane vesicles with a voltage difference across the membrane, i.e., an applied electric field in the membrane. Hyperpolarizing voltages displace S4 in such a manner as to sterically occlude the PIP2-binding site. Thus, in KCNQ1, the voltage sensor acts primarily as a regulator of PIP2 binding. The voltage sensors' influence on the channel's gate is indirect through the reaction sequence: voltage sensor movement â alter PIP2 ligand affinity â alter pore opening.
Assuntos
Canal de Potássio KCNQ1 , Lipídeos , Humanos , Canal de Potássio KCNQ1/metabolismo , Domínios Proteicos , Sítios de Ligação , Potenciais de AçãoRESUMO
KCNQ1, the major component of the slow-delayed rectifier potassium channel, is responsible for repolarization of cardiac action potential. Mutations in this channel can lead to a variety of diseases, most notably long QT syndrome. It is currently unknown how many of these mutations change channel function and structure on a molecular level. Since tetramerization is key to proper function and structure of the channel, it is likely that mutations modify the stability of KCNQ1 oligomers. Presently, the C-terminal domain of KCNQ1 has been noted as the driving force for oligomer formation. However, truncated versions of this protein lacking the C-terminal domain still tetramerize. Therefore, we explored the role of native cysteine residues in a truncated construct of human KCNQ1, amino acids 100-370, by blocking potential interactions of cysteines with a nitroxide based spin label. Mobility of the spin labels was investigated with continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. The oligomerization state was examined by gel electrophoresis. The data provide information on tetramerization of human KCNQ1 without the C-terminal domain. Specifically, how blocking the side chains of native cysteines residues reduces oligomerization. A better understanding of tetramer formation could provide improved understanding of the molecular etiology of long QT syndrome and other diseases related to KCNQ1.
Assuntos
Síndrome do QT Longo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Cisteína/genética , Mutação , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismoRESUMO
The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and ß-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and ß-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking ß-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.
Assuntos
Canal de Potássio KCNQ1 , Nó Sinoatrial , Animais , Humanos , Nó Sinoatrial/metabolismo , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Colforsina/farmacologia , Células HEK293 , Adrenérgicos , Potenciais de Ação/fisiologiaRESUMO
The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a known regulator of membrane protein trafficking. PI(3,5)P2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P2 binding sites, the known PIP2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P2 to both sites.