RESUMEN
OBJECTIVE: Mutations of the KCNQ2 gene, which encodes the Kv 7.2 subunit of voltage-gated M-type potassium channels, have been associated with epilepsy in the neonatal period. This developmental stage is unique in that the neurotransmitter gamma aminobutyric acid (GABA), which is inhibitory in adults, triggers excitatory action due to a reversed chloride gradient. METHODS: To examine whether KCNQ2-related neuronal hyperexcitability involves neonatally excitatory GABA, we examined 1-week-old knockin mice expressing the Kv 7.2 variant p.Tyr284Cys (Y284C). RESULTS: Brain slice electrophysiology revealed elevated CA1 hippocampal GABAergic interneuron activity with respect to presynaptic firing and postsynaptic current frequency. Blockade with the GABAA receptor antagonist bicuculline decreased ictal-like bursting in brain slices with lowered divalent ion concentration, which is consistent with GABA mediating an excitatory function that contributes to the hyperexcitability observed in mutant animals. SIGNIFICANCE: We conclude that excitatory GABA contributes to the phenotype in these animals, which raises the question of whether this special type of neurotransmission has broader importance in neonatal epilepsy than is currently recognized.
Asunto(s)
Epilepsia/genética , Epilepsia/metabolismo , Canal de Potasio KCNQ2/genética , Mutación/genética , Proteínas del Tejido Nervioso/genética , Transmisión Sináptica/genética , Ácido gamma-Aminobutírico/metabolismo , Animales , Animales Recién Nacidos , Biofisica , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Modelos Animales de Enfermedad , Estimulación Eléctrica , GABAérgicos/farmacología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Interneuronas/efectos de los fármacos , Interneuronas/fisiología , Magnesio/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
OBJECTIVE: To describe the unique phenotype and genetic findings in a 57-year-old female with a rare form of congenital myasthenic syndrome (CMS) associated with longstanding muscle fatigability, and to investigate the underlying pathophysiology. METHODS: We used whole-cell voltage clamping to compare the biophysical parameters of wild-type and Arg1457His-mutant Nav 1.4. RESULTS: Clinical and neurophysiological evaluation revealed features consistent with CMS. Sequencing of candidate genes indicated no abnormalities. However, analysis of SCN4A, the gene encoding the skeletal muscle sodium channel Nav 1.4, revealed a homozygous mutation predicting an arginine-to-histidine substitution at position 1457 (Arg1457His), which maps to the channel's voltage sensor, specifically D4/S4. Whole-cell patch clamp studies revealed that the mutant required longer hyperpolarization to recover from fast inactivation, which produced a profound use-dependent current attenuation not seen in the wild type. The mutant channel also had a marked hyperpolarizing shift in its voltage dependence of inactivation as well as slowed inactivation kinetics. INTERPRETATION: We conclude that Arg1457His compromises muscle fiber excitability. The mutant fast-inactivates with significantly less depolarization, and it recovers only after extended hyperpolarization. The resulting enhancement in its use dependence reduces channel availability, which explains the patient's muscle fatigability. Arg1457His offers molecular insight into a rare form of CMS precipitated by sodium channel inactivation defects. Given this channel's involvement in other muscle disorders such as paramyotonia congenita and hyperkalemic periodic paralysis, our study exemplifies how variations within the same gene can give rise to multiple distinct dysfunctions and phenotypes, revealing residues important in basic channel function.
Asunto(s)
Síndromes Miasténicos Congénitos/diagnóstico , Síndromes Miasténicos Congénitos/genética , Canal de Sodio Activado por Voltaje NAV1.4/genética , Recuperación de la Función/genética , Secuencia de Aminoácidos , Femenino , Células HEK293 , Humanos , Activación del Canal Iónico/genética , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
On April 21, 2015, the first SCN8A Encephalopathy Research Group convened in Washington, DC, to assess current research into clinical and pathogenic features of the disorder and prepare an agenda for future research collaborations. The group comprised clinical and basic scientists and representatives of patient advocacy groups. SCN8A encephalopathy is a rare disorder caused by de novo missense mutations of the sodium channel gene SCN8A, which encodes the neuronal sodium channel Nav 1.6. Since the initial description in 2012, approximately 140 affected individuals have been reported in publications or by SCN8A family groups. As a result, an understanding of the severe impact of SCN8A mutations is beginning to emerge. Defining a genetic epilepsy syndrome goes beyond identification of molecular etiology. Topics discussed at this meeting included (1) comparison between mutations of SCN8A and the SCN1A mutations in Dravet syndrome, (2) biophysical properties of the Nav 1.6 channel, (3) electrophysiologic effects of patient mutations on channel properties, (4) cell and animal models of SCN8A encephalopathy, (5) drug screening strategies, (6) the phenotypic spectrum of SCN8A encephalopathy, and (7) efforts to develop a bioregistry. A panel discussion of gaps in bioregistry, biobanking, and clinical outcomes data was followed by a planning session for improved integration of clinical and basic science research. Although SCN8A encephalopathy was identified only recently, there has been rapid progress in functional analysis and phenotypic classification. The focus is now shifting from identification of the underlying molecular cause to the development of strategies for drug screening and prioritized patient care.
Asunto(s)
Encefalopatías/genética , Epilepsia/etiología , Epilepsia/genética , Canal de Sodio Activado por Voltaje NAV1.6/genética , Simbiosis/genética , Animales , Anticonvulsivantes/uso terapéutico , Encefalopatías/complicaciones , Encefalopatías/tratamiento farmacológico , Progresión de la Enfermedad , Evaluación Preclínica de Medicamentos , Epilepsias Mioclónicas/tratamiento farmacológico , Epilepsias Mioclónicas/genética , Epilepsia/tratamiento farmacológico , Humanos , Modelos Moleculares , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , FenotipoRESUMEN
Cardiac toxicity represents one of the leading causes of drug failure along different stages of drug development. Multiple very successful pharmaceuticals had to be pulled from the market or labeled with strict usage warnings due to adverse cardiac effects. In order to protect clinical trial participants and patients, the International Conference on Harmonization published guidelines to recommend that all new drugs to be tested preclinically for hERG (Kv11.1) channel sensitivity before submitting for regulatory reviews. However, extensive studies have demonstrated that measurement of hERG activity has limitations due to the multiple molecular targets of drug compound through which it may mitigate or abolish a potential arrhythmia, and therefore, a model measuring multiple ion channel effects is likely to be more predictive. Several phenotypic rapid-throughput methods have been developed to predict the potential cardiac toxic compounds in the early stages of drug development using embryonic stem cells- or human induced pluripotent stem cell-derived cardiomyocytes. These rapid-throughput methods include microelectrode array-based field potential assay, impedance-based or Ca(2+) dynamics-based cardiomyocytes contractility assays. This review aims to discuss advantages and limitations of these phenotypic assays for cardiac toxicity assessment.
Asunto(s)
Fármacos Cardiovasculares/toxicidad , Evaluación Preclínica de Medicamentos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Cardiotoxicidad , Células Cultivadas , Canal de Potasio ERG1/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Humanos , Microelectrodos , Retirada de Medicamento por SeguridadRESUMEN
Benzodiazepines are the current first-line standard-of-care treatment for status epilepticus but fail to terminate seizures in about one third of cases. Synaptic GABAA receptors, which mediate phasic inhibition in central circuits, are the molecular target of benzodiazepines. As status epilepticus progresses, these receptors are internalized and become functionally inactivated, conferring benzodiazepine resistance, which is believed to be a major cause of treatment failure. GABAA receptor positive allosteric modulator neuroactive steroids, such as allopregnanolone, also potentiate synaptic GABAA receptors, but in addition they enhance extrasynaptic GABAA receptors that mediate tonic inhibition. Extrasynaptic GABAA receptors are not internalized, and desensitization of these receptors does not occur during continuous seizures in status epilepticus models. Here we review the broad-spectrum antiseizure activity of allopregnanolone in animal seizure models and the evidence for its activity in models of status epilepticus. We also demonstrate that allopregnanolone inhibits ongoing behavioral and electrographic seizures in a model of status epilepticus, even when there is benzodiazepine resistance. Parenteral allopregnanolone may provide an improved treatment for refractory status epilepticus.
Asunto(s)
Anestésicos/uso terapéutico , Estado Epiléptico/tratamiento farmacológico , Animales , Benzodiazepinas/metabolismo , Diazepam/farmacología , Humanos , Neurotransmisores/uso terapéutico , Pregnanolona/farmacologíaRESUMEN
Myotonia is a heritable disorder in which patients are unable to willfully relax their muscles. The physiological basis for myotonia lies in well-established deficiencies of skeletal muscle chloride and sodium conductances. What is unclear is how normal muscle function can temporarily return with repeated movement, the so-called "warm-up" phenomenon. Electrophysiological analyses of the skeletal muscle voltage-gated sodium channel Nav 1.4 (gene name SCN4A), a key player in myotonia, have revealed several parallels between the Nav 1.4 biophysical signature, specifically slow-inactivation, and myotonic warm-up, which suggest that Nav 1.4 is critical not only in producing the myotonic reaction, but also in mediating the warm-up.
Asunto(s)
Fibras Musculares Esqueléticas/metabolismo , Miotonía/metabolismo , Trastornos Miotónicos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Canales de Cloruro/metabolismo , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Miotonía/fisiopatología , Trastornos Miotónicos/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.4/genéticaRESUMEN
Ion channels, specifically voltage-gated sodium channels (Na(v)s), are common culprits in inheritable seizure disorders. Some Na(v) isoforms are particularly susceptible, while others are only weakly associated with neuronal hyperexcitability. Representative of the latter group is Na(v)1.2 (gene name SCN2A): despite its abundance in the brain, Na(v)1.2-related epilepsy is rare and only few studies have been conducted as to the pathophysiological basis of Na(v)1.2 in neuronal hyperexcitability. We here present a detailed functional analysis of Na(v)1.2 mutant, R1312T, which was originally found in a child with Dravet syndrome (formerly known as severe myoclonic epilepsy of infancy or SMEI). Whole-cell voltage clamp analysis revealed clearly compromised function: the mutant channels fast- and slow-inactivated at markedly more negative potentials and recovered from fast inactivation more slowly, which resulted in a use-dependent current reduction to less than 50% of wildtype levels. We also noted a small hyperpolarizing shift in the voltage dependence of activation. Our findings expand the spectrum of abnormal Na(v) channel behavior in epilepsy and raise the question as to how loss-of-function in a sodium channel predominantly expressed in excitatory neurons can lead to hyperexcitability.
Asunto(s)
Arginina/genética , Fenómenos Biofísicos/genética , Epilepsias Mioclónicas/genética , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.2/genética , Treonina/genética , Estimulación Eléctrica , Células HEK293 , Humanos , Activación del Canal Iónico/genética , Potenciales de la Membrana/genética , Mutagénesis Sitio-Dirigida/métodos , Técnicas de Placa-Clamp , Factores de Tiempo , TransfecciónRESUMEN
Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.
Asunto(s)
Epilepsia Generalizada/genética , Proteínas del Tejido Nervioso/genética , Canales de Sodio/genética , Células Cultivadas , Clonación Molecular , Humanos , Activación del Canal Iónico/genética , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.1 , Proteínas Recombinantes/genéticaRESUMEN
Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel alpha-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is approximately 0.3 ms in the -30 to -10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.
Asunto(s)
Epilepsia Generalizada/genética , Epilepsia Generalizada/fisiopatología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Canales de Sodio/genética , Canales de Sodio/fisiología , Alelos , Línea Celular , Membrana Celular/química , Membrana Celular/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Potenciales de la Membrana/fisiología , Mutación , Canal de Sodio Activado por Voltaje NAV1.1 , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Neuronas/fisiología , Técnicas de Placa-Clamp , Fenotipo , Proteínas Recombinantes/genética , Convulsiones Febriles/genética , Convulsiones Febriles/fisiopatología , Canales de Sodio/análisisRESUMEN
The inwardly rectifying K+ (Kir) channel Kir4.1 is responsible for astroglial K+ buffering. We recently found that tricyclic antidepressants (TCAs) inhibit Kir4.1 channel currents, which suggests that astroglial Kir currents might be involved in the pharmacological action of antidepressants. We therefore further examined the effects of the currently most popular antidepressants, selective serotonin reuptake inhibitors (SSRIs), and other related agents on Kir4.1 channels heterologously expressed in HEK293T cells. The whole-cell patch clamp technique was used. Fluoxetine, the typical SSRI, inhibited Kir4.1 channel currents in a concentration-dependent manner with an IC50 value of 15.2 microM. The inhibitory effect of fluoxetine was reversible and essentially voltage-independent. Fluoxetine had little or no effect upon Kir1.1 (ROMK1) or Kir2.1 (IRK1) channel currents. Other SSRIs, sertraline and fluvoxamine, also inhibited Kir4.1 channel currents whereas the tetracyclic (mianserin) or the 5-HT1A receptor-related (buspirone) antidepressants did not. This study shows that SSRIs such as fluoxetine and sertraline preferentially block astroglial Kir4.1 rather than Kir1.1 or Kir2.1 channels in the brain, which may be implicated in their therapeutic and/or adverse actions.
Asunto(s)
Astrocitos/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio de Rectificación Interna/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Antidepresivos Tricíclicos/farmacología , Astrocitos/efectos de los fármacos , Células Cultivadas , Interpretación Estadística de Datos , Electrofisiología , Fluoxetina/farmacología , Humanos , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/efectos de los fármacos , TransfecciónRESUMEN
Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha1 subunit (NaV1.1), are associated with at least two forms of epilepsy, generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). We examined the functional properties of four GEFS+ alleles and one SMEI allele using whole-cell patch-clamp analysis of heterologously expressed recombinant human SCN1A. One previously reported GEFS+ mutation (I1656M) and an additional novel allele (R1657C), both affecting residues in a voltage-sensing S4 segment, exhibited a similar depolarizing shift in the voltage dependence of activation. Additionally, R1657C showed a 50% reduction in current density and accelerated recovery from slow inactivation. Unlike three other GEFS+ alleles that we recently characterized, neither R1657C nor I1656M gave rise to a persistent, noninactivating current. In contrast, two other GEFS+ mutations (A1685V and V1353L) and L986F, an SMEI-associated allele, exhibited complete loss of function. In conclusion, our data provide evidence for a wide spectrum of sodium channel dysfunction in familial epilepsy and demonstrate that both GEFS+ and SMEI can be associated with nonfunctional SCN1A alleles.
Asunto(s)
Epilepsia/genética , Epilepsia/fisiopatología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Canales de Sodio/genética , Canales de Sodio/fisiología , Conductividad Eléctrica , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Predisposición Genética a la Enfermedad , Humanos , Activación del Canal Iónico , Cinética , Masculino , Mutación , Mutación Missense , Canal de Sodio Activado por Voltaje NAV1.1 , Neuronas/fisiología , Técnicas de Placa-Clamp , Convulsiones Febriles/genética , Convulsiones Febriles/fisiopatologíaRESUMEN
Research involving recombinant voltage-gated sodium (Nav) channels has unique challenges. Multiple factors contribute, but undoubtedly at the top of the list is these channels' DNA instability. Once introduced into bacterial hosts, Nav channel plasmid DNA will almost invariably emerge mutagenized and unusable, unless special conditions are adopted. This is particularly true for Nav1.1 (gene name SCN1A), Nav1.2 (SCN2A), and Nav1.6 (SCN8A), but less so for Nav1.4 (SCN4A) and Nav1.5 (SCN5A) while other Nav channel isoforms such as Nav1.7 (SCN9A) lie in between. The following recommendations for Nav plasmid DNA amplification and preparation address this problem. Three points are essential:â¢Bacterial propagation using Stbl2 cells at or below 30 °C.â¢Bias toward slow-growing, small bacterial colonies.â¢Comprehensive sequencing of the entire Nav channel coding region.
RESUMEN
Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.
Asunto(s)
Técnicas de Sustitución del Gen/métodos , Canal de Potasio KCNQ2/genética , Animales , Conducta Animal , Células Madre Embrionarias/metabolismo , Epilepsia Benigna Neonatal/inducido químicamente , Epilepsia Benigna Neonatal/genética , Epilepsia Benigna Neonatal/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Mutación , Pentilenotetrazol/efectos adversos , Embarazo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de TiempoRESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.
Asunto(s)
Exones , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Mutación , Canales de Potasio/genética , Convulsiones Febriles/genética , Secuencia de Aminoácidos , Animales , Estudios de Casos y Controles , Niño , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Cinética , Potenciales de la Membrana , Ratones , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Linaje , Canales de Potasio/metabolismo , Ratas , Convulsiones Febriles/metabolismo , Convulsiones Febriles/patología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
Human skeletal muscle channelopathies (HSMCs) are a group of heritable conditions with ion channel-related etiology and similar presentation. To create a comprehensive picture of the phenotypic spectrum for each condition and to devise a strategy that facilitates the differential diagnosis, we collected the genotype and phenotype information from more than 500 previously published HSMC studies. Using these records, we were able to identify clear correlations between particular clinical features and the underlying alteration(s) in the genes SCN4A, CACNA1S, KCNJ2, and CLCN1. This allowed us to develop a clinical, symptom-based, binary decision flow algorithm that predicts the proper genetic origin with high accuracy (0.88-0.93). The algorithm was implemented in a stand-alone online tool ("CGPS"- http://cgps.ddd.co.kr ) to assist with HSCM diagnosis in the clinical practice. The CGPS provides simple, symptom-oriented navigation that guides the user to the most likely molecular basis of the presentation, which permits highly targeted genetic screens and, upon confirmation, tailored pharmacotherapy based on the molecular origin.
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Canalopatías/genética , Músculo Esquelético/patología , Enfermedades Musculares/genética , Algoritmos , Inteligencia Artificial , Canalopatías/patología , Pruebas Genéticas , Genotipo , Humanos , Enfermedades Musculares/patología , Mutación , FenotipoRESUMEN
BACKGROUND: Dravet syndrome is a devastating infantile-onset epilepsy syndrome with cognitive deficits and autistic traits caused by genetic alterations in SCN1A gene encoding the α-subunit of the voltage-gated sodium channel Na(v)1.1. Disease modeling using patient-derived induced pluripotent stem cells (iPSCs) can be a powerful tool to reproduce this syndrome's human pathology. However, no such effort has been reported to date. We here report a cellular model for DS that utilizes patient-derived iPSCs. RESULTS: We generated iPSCs from a Dravet syndrome patient with a c.4933C>T substitution in SCN1A, which is predicted to result in truncation in the fourth homologous domain of the protein (p.R1645*). Neurons derived from these iPSCs were primarily GABAergic (>50%), although glutamatergic neurons were observed as a minor population (<1%). Current-clamp analyses revealed significant impairment in action potential generation when strong depolarizing currents were injected. CONCLUSIONS: Our results indicate a functional decline in Dravet neurons, especially in the GABAergic subtype, which supports previous findings in murine disease models, where loss-of-function in GABAergic inhibition appears to be a main driver in epileptogenesis. Our data indicate that patient-derived iPSCs may serve as a new and powerful research platform for genetic disorders, including the epilepsies.
Asunto(s)
Epilepsias Mioclónicas/patología , Células Madre Pluripotentes Inducidas/patología , Modelos Biológicos , Potenciales de Acción , Adulto , Diferenciación Celular , Epilepsias Mioclónicas/fisiopatología , Femenino , Genes Reporteros/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Lentivirus/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Neuronas/metabolismo , Neuronas/patología , Parvalbúminas/genética , Parvalbúminas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto JovenRESUMEN
Propofol is a rapidly acting water-insoluble non-barbiturate anesthetic agent that is widely used as an intravenous sedative-hypnotic agent. Anecdotal evidence indicates that propofol may be effective at terminating intractable migraine headache. Cortical spreading depression (CSD) is believed to be the neural correlate of migraine aura and may be a trigger for migraine pain. Agents that block the induction or slow the spread of CSD may be of utility in treating migraine. Here we examined the ability of propofol hemisuccinate (PHS), a water-soluble prodrug of propofol, to affect CSD in mice. For comparison, we examinined dizocilpine, an NMDA receptor antagonist, that is well recognized to inhibit CSD. When administered 15min prior to activation of CSD by KCl application to the cortex, intraperitoneal PHS at doses of 120 and 200mg/kg decreased the number of CSD deflections without an effect on CSD amplitude, and at 200mg/kg caused a 77% reduction in CSD velocity. The minimally-effective dose of PHS (120mg/kg) did not cause sedation or motor impairment and while some animals receiving 200mg/kg did demonstrate motor impariment none exhibited loss-of-righting reflex (anesthesia). Dizocilpine produced comparable inhibition of CSD at doses of 0.5 and 2.5mg/kg. We conclude that acute PHS treatment inhibits CSD. Our results indicate that propofol, or its prodrug PHS, are worthy of further investigation as a treatment for migraine.
Asunto(s)
Anestésicos/farmacología , Corteza Cerebral/efectos de los fármacos , Depresión de Propagación Cortical/efectos de los fármacos , Profármacos/farmacología , Propofol/farmacología , Anestésicos/uso terapéutico , Animales , Masculino , Ratones , Trastornos Migrañosos/tratamiento farmacológico , Actividad Motora/efectos de los fármacos , Profármacos/uso terapéutico , Propofol/uso terapéutico , Reflejo de Enderezamiento/efectos de los fármacosRESUMEN
OBJECTIVE: To electrophysiologically characterize the Na(v)1.4 mutant N440K found in a Korean family with a syndrome combining symptoms of paramyotonia congenita, hyperkalemic periodic paralysis, and potassium-aggravated myotonia. METHODS: We characterized transiently expressed wild-type and mutant Na(v)1.4 using whole-cell voltage-clamp analysis. RESULTS: N440K produced a significant depolarizing shift in the voltage dependence of fast inactivation and increased persistent current and acceleration in fast inactivation recovery, which gave rise to a 2-fold elevation in the dynamic availability of the mutant channels. In addition, the mutant channels required substantially longer and stronger depolarization to enter the slow-inactivated state. CONCLUSIONS: N440K causes a gain of function consistent with skeletal muscle hyperexcitability as observed in individuals with the mutation. How the same mutation results in distinct phenotypes in the 2 kindreds remains to be determined.
Asunto(s)
Activación del Canal Iónico/genética , Potenciales de la Membrana/genética , Trastornos Miotónicos/fisiopatología , Canal de Sodio Activado por Voltaje NAV1.4/genética , Parálisis Periódica Hiperpotasémica/fisiopatología , Adolescente , Adulto , Electromiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/fisiopatología , Mutación , Trastornos Miotónicos/genética , Parálisis Periódica Hiperpotasémica/genéticaRESUMEN
Over the past 10 years mutations in voltage-gated sodium channels (Na(v)s) have become closely associated with inheritable forms of epilepsy. One isoform in particular, Na(v)1.1 (gene symbol SCN1A), appears to be a superculprit, registering with more than 330 mutations to date. The associated phenotypes range from benign febrile seizures to extremely serious conditions, such as Dravet's syndrome (SMEI). Despite the wealth of information, mutational analyses are cumbersome, owing to inconsistencies among the Na(v)1.1 sequences to which different research groups refer. Splicing variability is the core problem: Na(v)1.1 co-exists in three isoforms, two of them lack 11 or 28 amino acids compared to full-length Na(v).1.1. This review establishes a standardized nomenclature for Na(v)1.1 variants so as to provide a platform from which future mutation analyses can be started without need for up-front data normalization. An online resource--SCN1A infobase--is introduced.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Canales de Sodio/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Bases de Datos Genéticas , Epilepsia/genética , Epilepsia/fisiopatología , Genotipo , Humanos , Datos de Secuencia Molecular , Herencia Multifactorial , Mutación , Canal de Sodio Activado por Voltaje NAV1.1 , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/clasificación , Fenotipo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Análisis de Secuencia de Proteína , Canales de Sodio/química , Canales de Sodio/clasificación , Terminología como AsuntoRESUMEN
Myotonia is a symptom of many different acquired and genetic muscular conditions that impair the relaxation phase of muscular contraction. Myotonia congenita is a specific inherited disorder of muscle membrane hyperexcitability caused by reduced sarcolemmal chloride conductance due to mutations in CLCN1, the gene coding for the main skeletal muscle chloride channel ClC-1. The disorder may be transmitted as either an autosomal-dominant or recessive trait with close to 130 currently known mutations. Although this is a rare disorder, elucidation of the pathophysiology underlying myotonia congenita established the importance of sarcolemmal chloride conductance in the control of muscle excitability and demonstrated the first example of human disease associated with the ClC family of chloride transporting proteins.