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1.
Neurobiol Dis ; 125: 31-44, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659983

RESUMO

SCN1A (NaV1.1 sodium channel) mutations cause Dravet syndrome (DS) and GEFS+ (which is in general milder), and are risk factors in other epilepsies. Phenotypic variability limits precision medicine in epilepsy, and it is important to identify factors that set phenotype severity and their mechanisms. It is not yet clear whether SCN1A mutations are necessary for the development of severe phenotypes or just for promoting seizures. A relevant example is the pleiotropic R1648H mutation that can cause either mild GEFS+ or severe DS. We used a R1648H knock-in mouse model (Scn1aRH/+) with mild/asymptomatic phenotype to dissociate the effects of seizures and of the mutation per se. The induction of short repeated seizures, at the age of disease onset for Scn1a mouse models (P21), had no effect in WT mice, but transformed the mild/asymptomatic phenotype of Scn1aRH/+ mice into a severe DS-like phenotype, including frequent spontaneous seizures and cognitive/behavioral deficits. In these mice, we found no major modifications in cytoarchitecture or neuronal death, but increased excitability of hippocampal granule cells, consistent with a pathological remodeling. Therefore, we demonstrate for our model that an SCN1A mutation is a prerequisite for a long term deleterious effect of seizures on the brain, indicating a clear interaction between seizures and the mutation for the development of a severe phenotype generated by pathological remodeling. Applied to humans, this result suggests that genetic alterations, even if mild per se, may increase the risk of second hits to develop severe phenotypes.


Assuntos
Epilepsia/genética , Epilepsia/patologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Convulsões/genética , Convulsões/patologia , Animais , Técnicas de Introdução de Genes , Hipocampo/patologia , Camundongos , Mutação , Fenótipo
2.
Bioorg Med Chem Lett ; 26(20): 4919-4924, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27641472

RESUMO

TRESK (Twik RElated Spinal cord K+ channel) is a member of the Twin Pore Domain potassium channel (K2P) family responsible for regulating neuronal excitability in dorsal root ganglion (DRG) and trigeminal (TG) neurons, peripheral neurons involved in pain transmission. As channel opening causes an outward K+ current responsible for cell hyperpolarisation, TRESK represents a potentially interesting target for pain treatment. However, as no crystal structure exists for this protein, the mechanisms involved in the opening action of its ligands are still poorly understood, making the development of new potent and selective openers challenging. In this work we present a structure activity relationship (SAR) of the known TRESK opener flufenamic acid (FFA) and some derivatives, investigating the functional effects of chemical modifications to build a TRESK homology model to support the biological results. A plausible binding mode is proposed, providing the first predictive hypothesis of a human TRESK opener binding site.


Assuntos
Ácido Flufenâmico/química , Ácido Flufenâmico/farmacologia , Canais de Potássio/química , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Relação Estrutura-Atividade
4.
J Clin Invest ; 131(21)2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34491914

RESUMO

Spreading depolarizations (SDs) are involved in migraine, epilepsy, stroke, traumatic brain injury, and subarachnoid hemorrhage. However, the cellular origin and specific differential mechanisms are not clear. Increased glutamatergic activity is thought to be the key factor for generating cortical spreading depression (CSD), a pathological mechanism of migraine. Here, we show that acute pharmacological activation of NaV1.1 (the main Na+ channel of interneurons) or optogenetic-induced hyperactivity of GABAergic interneurons is sufficient to ignite CSD in the neocortex by spiking-generated extracellular K+ build-up. Neither GABAergic nor glutamatergic synaptic transmission were required for CSD initiation. CSD was not generated in other brain areas, suggesting that this is a neocortex-specific mechanism of CSD initiation. Gain-of-function mutations of NaV1.1 (SCN1A) cause familial hemiplegic migraine type-3 (FHM3), a subtype of migraine with aura, of which CSD is the neurophysiological correlate. Our results provide the mechanism linking NaV1.1 gain of function to CSD generation in FHM3. Thus, we reveal the key role of hyperactivity of GABAergic interneurons in a mechanism of CSD initiation, which is relevant as a pathological mechanism of Nav1.1 FHM3 mutations, and possibly also for other types of migraine and diseases in which SDs are involved.


Assuntos
Depressão Alastrante da Atividade Elétrica Cortical , Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Transtornos de Enxaqueca/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.1/metabolismo , Neocórtex/metabolismo , Animais , Neurônios GABAérgicos/patologia , Interneurônios/patologia , Camundongos , Camundongos Transgênicos , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/patologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Neocórtex/patologia
6.
Neuropharmacology ; 55(1): 94-105, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18547595

RESUMO

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9+/-0.5 Hz. Application of dopamine (30 microM) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0+/-0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 microM, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 microM, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 microM, seven cells), but unaffected by the D2 antagonists sulpiride (10 microM, seven cells) or eticlopride (10 microM, six cells). Using voltage ramps, dopamine induced an inward current of 69+/-9.4 pA at a holding potential of -60 mV (n=17). This current was accompanied by an increase in input conductance of 1.55+/-0.35 nS which reversed at -30.6+/-2.3 mV, an effect mimicked by SKF38393 (10 microM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I(h) blocker, ZD7288, indicating effects independent of I(h). The increase in conductance was blocked by SCH23390 (2 microM, n=4), mimicked by the activator of adenylyl cyclase forskolin (10 microM, n=7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 microM, n=6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.


Assuntos
Potenciais de Ação/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Ativação do Canal Iônico/fisiologia , Receptores Dopaminérgicos/fisiologia , Núcleo Subtalâmico/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Dopamina/farmacologia , Dopaminérgicos/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp/métodos , Picrotoxina/farmacologia , Sódio/farmacologia , Núcleo Subtalâmico/citologia , Núcleo Subtalâmico/efeitos dos fármacos , Tetrodotoxina/farmacologia
7.
Br J Pharmacol ; 175(12): 2272-2283, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29150838

RESUMO

BACKGROUND AND PURPOSE: TREK two-pore-domain potassium (K2P ) channels play a critical role in regulating the excitability of somatosensory nociceptive neurons and are important mediators of pain perception. An understanding of the roles of TREK channels in pain perception and, indeed, in other pathophysiological conditions, has been severely hampered by the lack of potent and/or selective activators and inhibitors. In this study, we describe a new, selective opener of TREK channels, GI-530159. EXPERIMENTAL APPROACH: The effect of GI-530159 on TREK channels was demonstrated using 86 Rb efflux assays, whole-cell and single-channel patch-clamp recordings from recombinant TREK channels. The expression of K2P 2.1 (TREK1), K2P 10.1 (TREK2) and K2P 4.1 (TRAAK) channels was determined using transcriptome analysis from single dorsal root ganglion (DRG) cells. Current-clamp recordings from cultured rat DRG neurons were used to measure the effect of GI-530159 on neuronal excitability. KEY RESULTS: For recombinant human TREK1 channels, GI-530159 had similar low EC50 values in Rb efflux experiments and electrophysiological recordings. It activated TREK2 channels, but it had no detectable action on TRAAK channels nor any significant effect on other K channels tested. Current-clamp recordings from cultured rat DRG neurones showed that application of GI-530159 at 1 µM resulted in a significant reduction in firing frequency and a small hyperpolarization of resting membrane potential. CONCLUSIONS AND IMPLICATIONS: This study provides pharmacological evidence for the presence of mechanosensitive TREK K2P channels in sensory neurones and suggests that development of selective K2P channel openers like GI-530159 could aid in the development of novel analgesic agents. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.


Assuntos
Gânglios Espinais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Canais de Potássio de Domínios Poros em Tandem/agonistas , Animais , Células CHO , Linhagem Celular , Cricetulus , Relação Dose-Resposta a Droga , Gânglios Espinais/metabolismo , Células HEK293 , Humanos , Estrutura Molecular , Neurônios/metabolismo , Ratos , Relação Estrutura-Atividade
8.
Nat Genet ; 50(1): 54-61, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29229984

RESUMO

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células Receptoras Sensoriais/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Cromatina/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Genotipagem , Humanos , Locos de Características Quantitativas , Splicing de RNA , Células Receptoras Sensoriais/citologia , Análise de Sequência de RNA , Análise de Célula Única
9.
PLoS One ; 11(10): e0164004, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27706253

RESUMO

Layer Vb pyramidal cells are the major output neurons of the neocortex and transmit the outcome of cortical columnar signal processing to distant target areas. At the same time they contribute to local tactile information processing by emitting recurrent axonal collaterals into the columnar microcircuitry. It is, however, not known how exactly the two types of pyramidal cells, called slender-tufted and thick-tufted, contribute to the local circuitry. Here, we investigated in the rat barrel cortex the detailed quantitative morphology of biocytin-filled layer Vb pyramidal cells in vitro, which were characterized for their intrinsic electrophysiology with special emphasis on their action potential firing pattern. Since we stained the same slices for cytochrome oxidase, we could also perform layer- and column-related analyses. Our results suggest that in layer Vb the unambiguous action potential firing patterns "regular spiking (RS)" and "repetitive burst spiking (RB)" (previously called intrinsically burst spiking) correlate well with a distinct morphology. RS pyramidal cells are somatodendritically of the slender-tufted type and possess numerous local intralaminar and intracolumnar axonal collaterals, mostly reaching layer I. By contrast, their transcolumnar projections are less well developed. The RB pyramidal cells are somatodendritically of the thick-tufted type and show only relatively sparse local axonal collaterals, which are preferentially emitted as long horizontal or oblique infragranular collaterals. However, contrary to many previous slice studies, a substantial number of these neurons also showed axonal collaterals reaching layer I. Thus, electrophysiologically defined pyramidal cells of layer Vb show an input and output pattern which suggests RS cells to be more "locally segregating" signal processors whereas RB cells seem to act more on a "global integrative" scale.


Assuntos
Potenciais de Ação , Axônios/fisiologia , Células Piramidais/fisiologia , Animais , Dendritos/fisiologia , Fenômenos Eletrofisiológicos , Masculino , Ratos
10.
Sci Transl Med ; 8(335): 335ra56, 2016 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-27099175

RESUMO

In common with other chronic pain conditions, there is an unmet clinical need in the treatment of inherited erythromelalgia (IEM). TheSCN9Agene encoding the sodium channel Nav1.7 expressed in the peripheral nervous system plays a critical role in IEM. A gain-of-function mutation in this sodium channel leads to aberrant sensory neuronal activity and extreme pain, particularly in response to heat. Five patients with IEM were treated with a new potent and selective compound that blocked the Nav1.7 sodium channel resulting in a decrease in heat-induced pain in most of the patients. We derived induced pluripotent stem cell (iPSC) lines from four of five subjects and produced sensory neurons that emulated the clinical phenotype of hyperexcitability and aberrant responses to heat stimuli. When we compared the severity of the clinical phenotype with the hyperexcitability of the iPSC-derived sensory neurons, we saw a trend toward a correlation for individual mutations. The in vitro IEM phenotype was sensitive to Nav1.7 blockers, including the clinical test agent. Given the importance of peripherally expressed sodium channels in many pain conditions, our approach may have broader utility for a wide range of pain and sensory conditions.


Assuntos
Eritromelalgia/tratamento farmacológico , Células-Tronco Pluripotentes Induzidas/citologia , Dor/tratamento farmacológico , Dor/metabolismo , Éteres Fenílicos/uso terapêutico , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Sulfonamidas/uso terapêutico , Adulto , Eritromelalgia/genética , Feminino , Humanos , Masculino , Mutação/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Células Receptoras Sensoriais/citologia
11.
Br J Pharmacol ; 172(10): 2654-70, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25625641

RESUMO

BACKGROUND AND PURPOSE: NaV 1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH: The inhibition of Nav 1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTS: PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 channels (IC50 : ∼10 µM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50 : ∼10-18 µM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav 1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONS: Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.


Assuntos
Nociceptividade/efeitos dos fármacos , Ácidos Picolínicos/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Administração Oral , Animais , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.8/efeitos dos fármacos , Medição da Dor/efeitos dos fármacos , Ácidos Picolínicos/administração & dosagem , Ácidos Picolínicos/farmacocinética , Ratos , Tetrodotoxina/antagonistas & inibidores , Tetrodotoxina/farmacologia
12.
J Neurobiol ; 66(13): 1475-88, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17013926

RESUMO

Agonists at G-protein-coupled receptors in neurons of the dorsal raphe nucleus (DRN) of knock-out mice devoid of the serotonin transporter (5-HTT(-/-)) exhibit lower efficacy to inhibit cellular discharge than in wild-type counterparts. Using patch-clamp whole-cell recordings, we found that a G-protein-gated inwardly rectifying potassium (GIRK) current is involved in the inhibition of spike discharge induced by 5-HT1A agonists (5-carboxamidotryptamine (5-CT) and (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT); 50 nM-30 microM) in both wild-type and 5-HTT(-/-) female and male mice. These effects were mimicked by 5'-guanylyl-imido-diphosphate (Gpp(NH)p; 400 microM) dialysis into cells with differences between genders. The 5-HTT(-/-) knock-out mutation reduced the current density induced by Gpp(NH)p in females but not in males. These data suggest that the decreased response of 5-HT1A receptors to agonists in 5-HTT(-/-) mutants reflects notably alteration in the coupling between G-proteins and GIRK channels in females but not in males. Accordingly, gender differences in central 5-HT neurotransmission appear to depend-at least in part-on sex-related variations in corresponding receptor-G protein signaling mechanisms.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Neurônios/fisiologia , Núcleos da Rafe/citologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Caracteres Sexuais , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica/métodos , Feminino , Guanilil Imidodifosfato/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/classificação , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Piperazinas/farmacologia , Piridinas/farmacologia , Serotonina/análogos & derivados , Serotonina/farmacologia , Serotoninérgicos/farmacologia , Fatores de Tempo
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