RESUMO
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
TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.
Assuntos
Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Anestésicos/farmacologia , Animais , Cristalografia por Raios X , Condutividade Elétrica , Feminino , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Xenopus laevisRESUMO
The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain-containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP-dependent binding and regulation of TREK-1 potassium channels. We show that TREK-1 binds the AC9:POPDC1 complex and copurifies in a POPDC1-dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP-independent, TREK-1 association with AC9 and POPDC1 is reduced upon stimulation of the ß-adrenergic receptor (ßAR). AC9 activity is required for ßAR reduction of TREK-1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK-1 currents. Finally, deletion of the gene-encoding AC9 (Adcy9) gives rise to bradycardia at rest and stress-induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK-1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK-1 to regulate heart rate control.
Assuntos
Adenilil Ciclases , Canais de Potássio , Adenilil Ciclases/genéticaRESUMO
KV1.5 channels are key players in the regulation of vascular tone and atrial excitability and their impairment is associated with cardiovascular diseases including pulmonary arterial hypertension (PAH) and atrial fibrillation (AF). Unfortunately, pharmacological strategies to improve KV1.5 channel function are missing. Herein, we aimed to study whether the chaperone sigma-1 receptor (S1R) is able to regulate these channels and represent a new strategy to enhance their function. By using different electrophysiological and molecular techniques in X. laevis oocytes and HEK293 cells, we demonstrate that S1R physically interacts with KV1.5 channels and regulate their expression and function. S1R induced a bimodal regulation of KV1.5 channel expression/activity, increasing it at low concentrations and decreasing it at high concentrations. Of note, S1R agonists (PRE084 and SKF10047) increased, whereas the S1R antagonist BD1047 decreased, KV1.5 expression and activity. Moreover, PRE084 markedly increased KV1.5 currents in pulmonary artery smooth muscle cells and attenuated vasoconstriction and proliferation in pulmonary arteries. We also show that both KV1.5 channels and S1R, at mRNA and protein levels, are clearly downregulated in samples from PAH and AF patients. Moreover, the expression of both genes showed a positive correlation. Finally, the ability of PRE084 to increase KV1.5 function was preserved under sustained hypoxic conditions, as an in vitro PAH model. Our study provides insight into the key role of S1R in modulating the expression and activity of KV1.5 channels and highlights the potential role of this chaperone as a novel pharmacological target for pathological conditions associated with KV1.5 channel dysfunction.
Assuntos
Fibrilação Atrial , Receptores sigma , Humanos , Células HEK293 , Pulmão/patologia , Artéria Pulmonar , Receptores sigma/metabolismo , Receptor Sigma-1RESUMO
The KCNQ1 gene encodes the α-subunit of the cardiac voltage-gated potassium (Kv) channel KCNQ1, also denoted as Kv7.1 or KvLQT1. The channel assembles with the ß-subunit KCNE1, also known as minK, to generate the slowly activating cardiac delayed rectifier current IKs, a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function variants in KCNQ1 cause the congenital Long QT1 (LQT1) syndrome, characterized by delayed cardiac repolarization and a QT interval prolongation in the surface electrocardiogram (ECG). Autosomal dominant loss-of-function variants in KCNQ1 result in the LQT syndrome called Romano-Ward syndrome (RWS), while autosomal recessive variants affecting function, lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. The aim of this study was the characterization of novel KCNQ1 variants identified in patients with RWS to widen the spectrum of known LQT1 variants, and improve the interpretation of the clinical relevance of variants in the KCNQ1 gene. We functionally characterized nine human KCNQ1 variants using the voltage-clamp technique in Xenopus laevis oocytes, from which we report seven novel variants. The functional data was taken as input to model surface ECGs, to subsequently compare the functional changes with the clinically observed QTc times, allowing a further interpretation of the severity of the different LQTS variants. We found that the electrophysiological properties of the variants correlate with the severity of the clinically diagnosed phenotype in most cases, however, not in all. Electrophysiological studies combined with in silico modelling approaches are valuable components for the interpretation of the pathogenicity of KCNQ1 variants, but assessing the clinical severity demands the consideration of other factors that are included, for example in the Schwartz score.
Assuntos
Síndrome de Jervell-Lange Nielsen , Síndrome de Romano-Ward , Humanos , Síndrome de Romano-Ward/genética , Canal de Potássio KCNQ1/genética , Síndrome de Jervell-Lange Nielsen/genética , Fenótipo , Eletrocardiografia , Mutação , Canais de Potássio KCNQ/genéticaRESUMO
The voltage-dependent L-type calcium channel isoform CaV1.2 is critically involved in many physiological processes, e.g., in cardiac action potential formation, electromechanical coupling and regulation of insulin secretion by beta cells. Gain-of-function mutations in the calcium voltage-gated channel subunit alpha 1 C (CACNA1C) gene, encoding the CaV1.2 α1-subunit, cause Timothy syndrome (TS), a multisystemic disorder that includes autism spectrum disorders and long QT (LQT) syndrome. Strikingly, TS patients frequently suffer from hypoglycemia of yet unproven origin. Using next-generation sequencing, we identified a novel heterozygous CACNA1C mutation in a patient with congenital hyperinsulinism (CHI) and associated hypoglycemic episodes. We characterized the electrophysiological phenotype of the mutated channel using voltage-clamp recordings and in silico action potential modeling experiments. The identified CaV1.2L566P mutation causes a mixed electrophysiological phenotype of gain- and loss-of-function effects. In silico action potential modeling supports that this mixed electrophysiological phenotype leads to a tissue-specific impact on beta cells compared to cardiomyocytes. Thus, CACNA1C variants may be associated with non-syndromic hyperinsulinemic hypoglycemia without long-QT syndrome, explained by very specific electrophysiological properties of the mutated channel. We discuss different biochemical characteristics and clinical impacts of hypoglycemia in the context of CACNA1C variants and show that these may be associated with significant morbidity for Timothy Syndrome patients. Our findings underline that the potential of hypoglycemia warrants careful attention in patients with CACNA1C variants, and such variants should be included in the differential diagnosis of non-syndromic congenital hyperinsulinism.
Assuntos
Hiperinsulinismo Congênito , Síndrome do QT Longo , Sindactilia , Transtorno Autístico , Canais de Cálcio Tipo L/genética , Hiperinsulinismo Congênito/genética , Humanos , Mutação , Sindactilia/diagnóstico , Sindactilia/genéticaRESUMO
Cav1.3 voltage-gated L-type calcium channels (LTCCs) are involved in cardiac pacemaking, hearing and hormone secretion, but are also expressed postsynaptically in neurons. So far, homozygous loss of function mutations in CACNA1D encoding the Cav1.3 α1-subunit are described in congenital sinus node dysfunction and deafness. In addition, germline mutations in CACNA1D have been linked to neurodevelopmental syndromes including epileptic seizures, autism, intellectual disability and primary hyperaldosteronism. Here, a three-generation family with a syndromal phenotype of sinus node dysfunction, idiopathic epilepsy and attention deficit hyperactivity disorder (ADHD) is investigated. Whole genome sequencing and functional heterologous expression studies were used to identify the disease-causing mechanisms in this novel syndromal disorder. We identified a heterozygous non-synonymous variant (p.Arg930His) in the CACNA1D gene that cosegregated with the combined clinical phenotype in an autosomal dominant manner. Functional heterologous expression studies showed that the CACNA1D variant induces isoform-specific alterations of Cav1.3 channel gating: a gain of ion channel function was observed in the brain-specific short CACNA1D isoform (Cav1.3S), whereas a loss of ion channel function was seen in the long (Cav1.3L) isoform. The combined gain-of-function (GOF) and loss-of-function (LOF) induced by the R930H variant are likely to be associated with the rare combined clinical and syndromal phenotypes in the family. The GOF in the Cav1.3S variant with high neuronal expression is likely to result in epilepsy, whereas the LOF in the long Cav1.3L variant results in sinus node dysfunction.
Assuntos
Canais de Cálcio Tipo L , Epilepsia , Síndrome do Nó Sinusal , Humanos , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Isoformas de Proteínas/metabolismo , Síndrome do Nó Sinusal/genética , Síndrome do Nó Sinusal/metabolismo , Sequenciamento do ExomaRESUMO
Potassium channels of the tandem of two-pore-domain (K2P) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels. Recently obtained crystal structures of K2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate K2P channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different K2P channel blockers and activators.
Assuntos
Doença do Sistema de Condução Cardíaco/tratamento farmacológico , Moduladores de Transporte de Membrana/farmacologia , Transtornos de Enxaqueca/tratamento farmacológico , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Sítios de Ligação , Doença do Sistema de Condução Cardíaco/genética , Doença do Sistema de Condução Cardíaco/metabolismo , Doença do Sistema de Condução Cardíaco/patologia , Expressão Gênica , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons , Ligantes , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/classificação , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/metabolismo , Transtornos de Enxaqueca/patologia , Simulação de Dinâmica Molecular , Especificidade de Órgãos , Canais de Potássio de Domínios Poros em Tandem/classificação , Canais de Potássio de Domínios Poros em Tandem/genética , Ligação Proteica , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de ProteínaRESUMO
KCNQ1 encodes the voltage-gated potassium (Kv) channel KCNQ1, also known as KvLQT1 or Kv7.1. Together with its ß-subunit KCNE1, also denoted as minK, this channel generates the slowly activating cardiac delayed rectifier current IKs, which is a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function mutations in KCNQ1 cause congenital long QT1 (LQT1) syndrome, characterized by a delayed cardiac repolarization and a prolonged QT interval in the surface electrocardiogram. Autosomal dominant loss-of-function mutations in KCNQ1 result in long QT syndrome, called Romano-Ward Syndrome (RWS), while autosomal recessive mutations lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. Here, we identified a homozygous KCNQ1 mutation, c.1892_1893insC (p.P631fs*20), in a patient with an isolated LQT syndrome (LQTS) without hearing loss. Nevertheless, the inheritance trait is autosomal recessive, with heterozygous family members being asymptomatic. The results of the electrophysiological characterization of the mutant, using voltage-clamp recordings in Xenopus laevis oocytes, are in agreement with an autosomal recessive disorder, since the IKs reduction was only observed in homomeric mutants, but not in heteromeric IKs channel complexes containing wild-type channel subunits. We found that KCNE1 rescues the KCNQ1 loss-of-function in mutant IKs channel complexes when they contain wild-type KCNQ1 subunits, as found in the heterozygous state. Action potential modellings confirmed that the recessive c.1892_1893insC LQT1 mutation only affects the APD of homozygous mutation carriers. Thus, our study provides the molecular mechanism for an atypical autosomal recessive LQT trait that lacks hearing impairment.
Assuntos
Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Síndrome de Romano-Ward/genética , Potenciais de Ação , Animais , Surdez/genética , Feminino , Genes Recessivos , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , Oócitos/fisiologia , Técnicas de Patch-Clamp , Linhagem , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Síndrome de Romano-Ward/etiologia , Xenopus laevisRESUMO
TASK channels belong to the two-pore-domain potassium (K2P) channels subfamily. These channels modulate cellular excitability, input resistance, and response to synaptic stimulation. TASK-channel inhibition led to membrane depolarization. TASK-3 is expressed in different cancer cell types and neurons. Thus, the discovery of novel TASK-3 inhibitors makes these bioactive compounds very appealing to explore new cancer and neurological therapies. TASK-3 channel blockers are very limited to date, and only a few heterofused compounds have been reported in the literature. In this article, we combined a pharmacophore hypothesis with molecular docking to address for the first time the rational design, synthesis, and evaluation of 5-(indol-2-yl)pyrazolo[3,4-b]pyridines as a novel family of human TASK-3 channel blockers. Representative compounds of the synthesized library were assessed against TASK-3 using Fluorometric imaging plate reader-Membrane Potential assay (FMP). Inhibitory properties were validated using two-electrode voltage-clamp (TEVC) methods. We identified one active hit compound (MM-3b) with our systematic pipeline, exhibiting an IC50 ≈ 30 µM. Molecular docking models suggest that compound MM-3b binds to TASK-3 at the bottom of the selectivity filter in the central cavity, similar to other described TASK-3 blockers such as A1899 and PK-THPP. Our in silico and experimental studies provide a new tool to predict and design novel TASK-3 channel blockers.
Assuntos
Simulação de Acoplamento Molecular , Bloqueadores dos Canais de Potássio , Canais de Potássio de Domínios Poros em Tandem , Piridinas , Humanos , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Piridinas/síntese química , Piridinas/químicaRESUMO
Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2W188â), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K2P) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2W188â causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2W188â knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2W188â loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK-1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.
Assuntos
Doença do Sistema de Condução Cardíaco/genética , Moléculas de Adesão Celular/genética , Predisposição Genética para Doença , Proteínas Musculares/genética , Potenciais de Ação , Animais , Bloqueio Atrioventricular/genética , Bradicardia/complicações , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Estudos de Associação Genética , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/patologia , Heterozigoto , Homozigoto , Humanos , Leucócitos/metabolismo , Camundongos Transgênicos , Proteínas Musculares/metabolismo , Mutação/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , RNA/metabolismo , Nó Sinoatrial/metabolismo , Estresse Fisiológico , Sequenciamento do Exoma , Xenopus laevisRESUMO
The hyperpolarization-activated cation current If is a key determinant for cardiac pacemaker activity. It is conducted by subunits of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family, of which HCN4 is predominant in mammalian heart. Both loss-of-function and gain-of-function mutations of the HCN4 gene are associated with sinus node dysfunction in humans; however, their functional impact is not fully understood yet. Here, we sought to characterize a HCN4 V759I variant detected in a patient with a family history of sick sinus syndrome. The genomic analysis yielded a mono-allelic HCN4 V759I variant in a 49-year-old woman presenting with a family history of sick sinus syndrome. This HCN4 variant was previously classified as putatively pathogenic because genetically linked to sudden infant death syndrome and malignant epilepsy. However, detailed electrophysiological and cell biological characterization of HCN4 V759I in Xenopus laevis oocytes and embryonic rat cardiomyocytes, respectively, did not reveal any obvious abnormality. Voltage dependence and kinetics of mutant channel activation, modulation of cAMP-gating by the neuronal HCN channel auxiliary subunit PEX5R, and cell surface expression were indistinguishable from wild-type HCN4. In good agreement, the clinically likewise affected mother of the patient does not exhibit the reported HCN4 variance. HCN4 V759I resembles an innocuous genetic HCN channel variant, which is not sufficient to disturb cardiac pacemaking. Once more, our work emphasizes the importance of careful functional interpretation of genetic findings not only in the context of hereditary cardiac arrhythmias.
Assuntos
Bradicardia/genética , Frequência Cardíaca , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Proteínas Musculares/genética , Mutação de Sentido Incorreto , Canais de Potássio/genética , Potenciais de Ação , Animais , Bradicardia/diagnóstico , Bradicardia/fisiopatologia , Células Cultivadas , Feminino , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Canais de Potássio/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , XenopusRESUMO
OBJECTIVE: The Popeye domain containing 3 (POPDC3) gene encodes a membrane protein involved in cyclic adenosine monophosphate (cAMP) signaling. Besides gastric cancer, no disease association has been described. We describe a new muscular dystrophy associated with this gene. METHODS: We screened 1,500 patients with unclassified limb girdle weakness or hyperCKemia for pathogenic POPDC3 variants. Five patients carrying POPDC3 variants were examined by muscle magnetic resonance imaging (MRI), muscle biopsy, and cardiac examination. We performed functional analyses in a zebrafish popdc3 knockdown model and heterologous expression of the mutant proteins in Xenopus laevis oocytes to measure TREK-1 current. RESULTS: We identified homozygous POPDC3 missense variants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) in 5 patients from 3 ethnically distinct families. Variants affected highly conserved residues in the Popeye (p.Leu155 and p.Leu217) and carboxy-terminal (p.Arg261) domains. The variants were almost absent from control populations. Probands' muscle biopsies were dystrophic, and serum creatine kinase levels were 1,050 to 9,200U/l. Muscle weakness was proximal with adulthood onset in most patients and affected lower earlier than upper limbs. Muscle MRI revealed fat replacement of paraspinal and proximal leg muscles; cardiac investigations were unremarkable. Knockdown of popdc3 in zebrafish, using 2 different splice-site blocking morpholinos, resulted in larvae with tail curling and dystrophic muscle features. All 3 mutants cloned in Xenopus oocytes caused an aberrant modulation of the mechano-gated potassium channel, TREK-1. INTERPRETATION: Our findings point to an important role of POPDC3 for skeletal muscle function and suggest that pathogenic variants in POPDC3 are responsible for a novel type of autosomal recessive limb girdle muscular dystrophy. ANN NEUROL 2019;86:832-843.
Assuntos
Moléculas de Adesão Celular/genética , Variação Genética/genética , Proteínas Musculares/genética , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/fisiologia , Distrofia Muscular do Cíngulo dos Membros/diagnóstico por imagem , Distrofia Muscular do Cíngulo dos Membros/genética , Adulto , Animais , Moléculas de Adesão Celular/química , Estudos de Coortes , Feminino , Técnicas de Silenciamento de Genes/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/química , Linhagem , Estrutura Secundária de Proteína , Xenopus laevis , Peixe-ZebraRESUMO
Complex neuropsychiatric-cardiac syndromes can be genetically determined. For the first time, the authors present a syndromal form of short QT syndrome in a 34-year-old German male patient with extracardiac features with predominant psychiatric manifestation, namely a severe form of secondary high-functioning autism spectrum disorder (ASD), along with affective and psychotic exacerbations, and severe dental enamel defects (with rapid wearing off his teeth) due to a heterozygous loss-of-function mutation in the CACNA1C gene (NM_000719.6: c.2399A > C; p.Lys800Thr). This mutation was found only once in control databases; the mutated lysine is located in the Cav1.2 calcium channel, is highly conserved during evolution, and is predicted to affect protein function by most pathogenicity prediction algorithms. L-type Cav1.2 calcium channels are widely expressed in the brain and heart. In the case presented, electrophysiological studies revealed a prominent reduction in the current amplitude without changes in the gating behavior of the Cav1.2 channel, most likely due to a trafficking defect. Due to the demonstrated loss of function, the p.Lys800Thr variant was finally classified as pathogenic (ACMG class 4 variant) and is likely to cause a newly described Cav1.2 channelopathy.
Assuntos
Arritmias Cardíacas , Transtorno Autístico , Canais de Cálcio Tipo L , Canalopatias , Esmalte Dentário , Mutação com Perda de Função , Transtornos do Humor , Adulto , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Canalopatias/genética , Canalopatias/metabolismo , Canalopatias/patologia , Esmalte Dentário/anormalidades , Esmalte Dentário/metabolismo , Esmalte Dentário/patologia , Humanos , Masculino , Transtornos do Humor/genética , Transtornos do Humor/metabolismo , Transtornos do Humor/patologiaRESUMO
BACKGROUND/AIMS: The two-pore-domain potassium channel TASK-1 regulates atrial action potential duration. Due to the atrium-specific expression of TASK-1 in the human heart and the functional upregulation of TASK-1 currents in atrial fibrillation (AF), TASK-1 represents a promising target for the treatment of AF. Therefore, detailed knowledge of the molecular determinants of TASK-1 inhibition may help to identify new drugs for the future therapy of AF. In the current study, the molecular determinants of TASK-1 inhibition by the potent and antiarrhythmic compound A293 (AVE1231) were studied in detail. METHODS: Alanine-scanning mutagenesis together with two-electrode voltage-clamp recordings were combined with in silico docking experiments. RESULTS: Here, we have identified Q126 located in the M2 segment together with L239 and N240 of the M4 segment as amino acids essential for the A293-mediated inhibition of TASK-1. These data indicate a binding site which is different to that of A1899 for which also residues of the pore signature sequence and the late M4 segments are essential. Using in silico docking experiments, we propose a binding site at the lower end of the cytosolic pore, located at the entry to lateral side fenestrations of TASK-1. Strikingly, TASK-1 inhibition by the low affinity antiarrhythmic TASK-1 blockers propafenone, amiodarone and carvedilol was also strongly diminished by mutations at this novel binding site. CONCLUSION: We have identified the A293 binding site in the central cavity of TASK-1 and propose that this site might represent a conserved site of action for many low affinity antiarrhythmic TASK-1 blockers.
Assuntos
Antiarrítmicos/química , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Xenopus laevisRESUMO
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels encode neuronal and cardiac pacemaker currents. The composition of pacemaker channel complexes in different tissues is poorly understood, and the presence of additional HCN modulating subunits was speculated. Here we show that vesicle-associated membrane protein-associated protein B (VAPB), previously associated with a familial form of amyotrophic lateral sclerosis 8, is an essential HCN1 and HCN2 modulator. VAPB significantly increases HCN2 currents and surface expression and has a major influence on the dendritic neuronal distribution of HCN2. Severe cardiac bradycardias in VAPB-deficient zebrafish and VAPB-/- mice highlight that VAPB physiologically serves to increase cardiac pacemaker currents. An altered T-wave morphology observed in the ECGs of VAPB-/- mice supports the recently proposed role of HCN channels for ventricular repolarization. The critical function of VAPB in native pacemaker channel complexes will be relevant for our understanding of cardiac arrhythmias and epilepsies, and provides an unexpected link between these diseases and amyotrophic lateral sclerosis.-Silbernagel, N., Walecki, M., Schäfer, M.-K. H., Kessler, M., Zobeiri, M., Rinné, S., Kiper, A. K., Komadowski, M. A., Vowinkel, K. S., Wemhöner, K., Fortmüller, L., Schewe, M., Dolga, A. M., Scekic-Zahirovic, J., Matschke, L. A., Culmsee, C., Baukrowitz, T., Monassier, L., Ullrich, N. D., Dupuis, L., Just, S., Budde, T., Fabritz, L., Decher, N. The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function.
Assuntos
Coração/fisiologia , Ativação do Canal Iônico , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Marca-Passo Artificial , Animais , Proteínas de Transporte/fisiologia , Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Feminino , Células HeLa , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , Camundongos Knockout , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Proteínas de Transporte Vesicular , Xenopus laevis , Peixe-ZebraRESUMO
RATIONALE: Familial sinus node and atrioventricular conduction dysfunction is a rare disorder that leads to paroxysmal dizziness, fatigue, and syncope because of a temporarily or permanently reduced heart rate. To date, only a few genes for familial sinus and atrioventricular conduction dysfunction are known, and the majority of cases remain pathogenically unresolved. OBJECTIVE: We aim to identify the disease gene in a large 3-generation family (n=25) with autosomal dominant sinus node dysfunction (SND) and atrioventricular block (AVB) and to characterize the mutation-related pathomechanisms in familial SND+AVB. METHODS AND RESULTS: Genome-wide linkage analysis mapped the SND+AVB disease locus to chromosome 7q21.1-q31.1 (2-point logarithm of the odds score: 4.64; θ=0); in this region, targeted exome sequencing identified a novel heterozygous mutation (p.Arg52Leu) in the GNB2 gene that strictly cosegregated with the SND+AVB phenotype. GNB2 encodes the ß2 subunit (Gß2) of the heterotrimeric G-protein complex that is being released from G-protein-coupled receptors on vagal stimulation. In 2 heterologous expression systems (HEK-293T cells and Xenopus laevis oocytes), an enhanced activation of the G-protein-activated K+ channel (GIRK; Kir3.1/Kir3.4) was shown when mutant Gß2 was coexpressed with Gγ2; this was in contrast to coexpression of mutant Gß2-Gγ2 with other cardiac ion channels (HCN4, HCN2, and Cav1.2). Molecular dynamics simulations suggested a reduced binding property of mutant Gß2 to cardiac GIRK channels when compared with native Gß2. CONCLUSIONS: A GNB2 gene mutation is associated with familial SND+AVB and leads to a sustained activation of cardiac GIRK channels, which is likely to hyperpolarize the myocellular membrane potential and thus reduces their spontaneous activity. Our findings describe for the first time a role of a mutant G-protein in the nonsyndromic pacemaker disease because of GIRK channel activation.
Assuntos
Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/fisiopatologia , Proteínas de Ligação ao GTP/genética , Mutação/genética , Síndrome do Nó Sinusal/genética , Síndrome do Nó Sinusal/fisiopatologia , Adulto , Sequência de Aminoácidos , Bloqueio Atrioventricular/diagnóstico , Feminino , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla/métodos , Células HEK293 , Sistema de Condução Cardíaco/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome do Nó Sinusal/diagnóstico , Nó Sinoatrial/fisiologia , Adulto JovemRESUMO
The physiological, intrinsic activity of noradrenergic locus coeruleus (LC) neurons is important for the control of sleep/wakefulness, cognition and autonomous body functions. Dysregulations of the LC-noradrenergic network contribute to the pathogenesis of psychiatric disorders and are key findings in early stages of neurodegenerative diseases. Therefore, identifying ion channels mediating the intrinsic pacemaking mechanism of LC neurons, which is in turn directly coupled to Ca2+ homeostasis and cell survival signaling pathways, can help to foster our understanding of the vulnerability of these neurons in neurodegenerative diseases. Small-conductance Ca2+-activated K+ (SK) channels regulate the intrinsic firing patterns in different central neurons and are essential regulators of the intracellular Ca2+ homeostasis. However, the role of SK channels for the intrinsic pacemaking of LC neurons in mice is still unclear. Therefore we performed qPCR expression analysis as well as patch clamp recordings of in vitro brainstem slices, for instance testing SK channel blockers and activators like apamin and NS309, respectively. Although we found a transcriptional expression of SK1, SK2 and SK3 channels, SK2 was the predominantly expressed subunit in mouse LC neurons. Using perforated-patch clamp experiments, we found that SK channels are essential regulators of the intrinsic pacemaking of LC neurons, mediating a large fraction of the afterhyperpolarization (AHP) in these cells. Consistent with a previous observation that a concerted action of L- and T-type Cav channels is essential for the pacemaking of LC neurons, we found that SK channel activation, and the respective AHP amplitude, is primarily coupled to Ca2+ influx via these types of Ca2+ channels. Our study identified SK2 channels as drug targets for the tuning of the pacemaker frequency in disorders involving a dysregulation of the LC.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Cálcio/metabolismo , Locus Cerúleo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Marca-Passo Artificial , Canais de Potássio/metabolismoRESUMO
TASK-3 is a two-pore domain potassium (K2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC50 = 56.8 ± 3.9 µM. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC50 = 14.2 ± 3.4 µM. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol.
Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Desenho de Fármacos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Bloqueadores dos Canais de Potássio/farmacocinéticaRESUMO
TASK-3 potassium (K+) channels are highly expressed in the central nervous system, regulating the membrane potential of excitable cells. TASK-3 is involved in neurotransmitter action and has been identified as an oncogenic K+ channel. For this reason, the understanding of the action mechanism of pharmacological modulators of these channels is essential to obtain new therapeutic strategies. In this study we describe the binding mode of the potent antagonist PK-THPP into the TASK-3 channel. PK-THPP blocks TASK-1, the closest relative channel of TASK-3, with almost nine-times less potency. Our results confirm that the binding is influenced by the fenestrations state of TASK-3 channels and occurs when they are open. The binding is mainly governed by hydrophobic contacts between the blocker and the residues of the binding site. These interactions occur not only for PK-THPP, but also for the antagonist series based on 5,6,7,8 tetrahydropyrido[4,3-d]pyrimidine scaffold (THPP series). However, the marked difference in the potency of THPP series compounds such as 20b, 21, 22 and 23 (PK-THPP) respect to compounds such as 17b, inhibiting TASK-3 channels in the micromolar range is due to the presence of a hydrogen bond acceptor group that can establish interactions with the threonines of the selectivity filter.