RESUMEN
Multiple voltage-gated Na+ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.
Asunto(s)
Antineoplásicos , Ensayos Analíticos de Alto Rendimiento/métodos , Mapas de Interacción de Proteínas/fisiología , Agonistas del Canal de Sodio Activado por Voltaje/aislamiento & purificación , Bloqueadores del Canal de Sodio Activado por Voltaje/aislamiento & purificación , Canales de Sodio Activados por Voltaje/efectos de los fármacos , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Factores de Crecimiento de Fibroblastos/agonistas , Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Factores de Crecimiento de Fibroblastos/química , Células HEK293 , Humanos , Complejos Multiproteicos/agonistas , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/química , Canal de Sodio Activado por Voltaje NAV1.6/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Unión Proteica , Agonistas del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Cortical hyperexcitability is an early and intrinsic feature of Amyotrophic Lateral Sclerosis (ALS), but the mechanisms underlying this critical neuronal dysfunction are poorly understood. Recently, we have demonstrated that layer V pyramidal neurons (PNs) in the primary motor cortex (M1) of one-month old (P30) G93A ALS mice display an early hyperexcitability status compared to Control mice. In order to investigate the time-dependent evolution of the cortical excitability in the G93A ALS model, here we have performed an electrophysiological and immunohistochemical study at three different mouse ages. M1 PNs from 14-days old (P14) G93A mice have shown no excitability alterations, while M1 PNs from 3-months old (P90) G93A mice have shown a hypoexcitability status, compared to Control mice. These age-dependent cortical excitability dysfunctions correlate with a similar time-dependent trend of the persistent sodium current (INaP) amplitude alterations, suggesting that INaP may play a crucial role in the G93A cortical excitability aberrations. Specifically, immunohistochemistry experiments have indicated that the expression level of the NaV1.6 channel, one of the voltage-gated Na+ channels mainly distributed within the central nervous system, varies in G93A primary motor cortex during disease progression, according to the excitability and INaP alterations, but not in other cortical areas. Microfluorometry experiments, combined with electrophysiological recordings, have verified that P30 G93A PNs hyperexcitability is associated to a greater accumulation of intracellular calcium ([Ca2+]i) compared to Control PNs, and that this difference is still present when G93A and Control PNs fire action potentials at the same frequency. These results suggest that [Ca2+]i de-regulation in G93A PNs may contribute to neuronal demise and that the NaV1.6 channels could be a potential therapeutic target to ameliorate ALS disease progression.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Corteza Motora/fisiopatología , Neuronas Motoras/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Potenciales de Acción/fisiología , Factores de Edad , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Calcio/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Corteza Motora/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genéticaRESUMEN
The present study was carried out to evaluate: the antiepileptic effect of dietary curcumin, and the effect of epileptic state and curcumin on the molecular expression of voltage-activated Na+ channel subtypes Nav1.1 and Nav1.6 in the iron-induced experimental epilepsy in the rat. Rats were divided into four groups; Group I (control rats), Group II (epileptic rats), Group III (curcumin-fed epileptic rats), and Group IV (curcumin-fed rats). Curcumin was fed chronically to rats approximately at the dose of 100 mg/kg body wt. The animals were made epileptic by intracortical injection of FeCl3. The mRNA and protein expressions of Nav1.1 and Nav1.6 were examined by RT-PCR analysis and immuno-histochemistry. Results showed a significant increase (upregulation) in the expression of both Nav1.1 and Nav1.6 with seizure activity in the cortex and hippocampus of epileptic rats. Epileptic rats fed with curcumin showed a marked decrease in epileptiform activity, and reduced mRNA and protein levels of Nav1.1. It appears that the antiepileptic action of curcumin may be associated with the downregulation of Nav1.1 in the cortex.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Curcumina/uso terapéutico , Epilepsia/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia/inducido químicamente , Hierro/toxicidad , Masculino , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.6/genética , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds' IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z' factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Potenciales de la Membrana/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Técnicas de Placa-Clamp/métodos , Bloqueadores de los Canales de Potasio/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Animales , Células CHO , Cricetulus , Evaluación Preclínica de Medicamentos , Expresión Génica , Ensayos Analíticos de Alto Rendimiento/instrumentación , Canal de Potasio Kv1.3/deficiencia , Canal de Potasio Kv1.3/genética , Canal de Sodio Activado por Voltaje NAV1.1/genética , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Canal de Sodio Activado por Voltaje NAV1.2/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.3/genética , Canal de Sodio Activado por Voltaje NAV1.3/metabolismo , Canal de Sodio Activado por Voltaje NAV1.4/genética , Canal de Sodio Activado por Voltaje NAV1.4/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/genética , Técnicas de Placa-Clamp/instrumentación , Cultivo Primario de Células , Ratas , Canales de Sodio/genética , Canales de Sodio/metabolismo , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , TransgenesRESUMEN
Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Red Nerviosa/metabolismo , Sinapsis/metabolismo , Tálamo/metabolismo , Animales , Modelos Animales de Enfermedad , Electroencefalografía/métodos , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/metabolismo , Ratones , Fenotipo , Convulsiones/genética , Convulsiones/metabolismoRESUMEN
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
BACKGROUND: NaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a "peripheral" sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, NaV1.2 and NaV1.6, display up-regulated expression in response to osmotic stress. RESULTS: Here we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress. CONCLUSIONS: NaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.