RESUMO
Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Potássio Shaw/metabolismo , Ataxias Espinocerebelares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Canais de Potássio Shaw/química , Canais de Potássio Shaw/genética , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Many rapidly firing neurons, including those in the medial nucleus of the trapezoid body (MNTB) in the auditory brain stem, express "high threshold" voltage-gated Kv3.1 potassium channels that activate only at positive potentials and are required for stimuli to generate rapid trains of actions potentials. We now describe the actions of two imidazolidinedione derivatives, AUT1 and AUT2, which modulate Kv3.1 channels. Using Chinese hamster ovary cells stably expressing rat Kv3.1 channels, we found that lower concentrations of these compounds shift the voltage of activation of Kv3.1 currents toward negative potentials, increasing currents evoked by depolarization from typical neuronal resting potentials. Single-channel recordings also showed that AUT1 shifted the open probability of Kv3.1 to more negative potentials. Higher concentrations of AUT2 also shifted inactivation to negative potentials. The effects of lower and higher concentrations could be mimicked in numerical simulations by increasing rates of activation and inactivation respectively, with no change in intrinsic voltage dependence. In brain slice recordings of mouse MNTB neurons, both AUT1 and AUT2 modulated firing rate at high rates of stimulation, a result predicted by numerical simulations. Our results suggest that pharmaceutical modulation of Kv3.1 currents represents a novel avenue for manipulation of neuronal excitability and has the potential for therapeutic benefit in the treatment of hearing disorders.
Assuntos
Tronco Encefálico/efeitos dos fármacos , Hidantoínas/farmacologia , Neurônios/efeitos dos fármacos , Neurotransmissores/farmacologia , Piridinas/farmacologia , Canais de Potássio Shaw/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Tronco Encefálico/fisiologia , Células CHO , Simulação por Computador , Cricetulus , Hidantoínas/química , Camundongos Endogâmicos C57BL , Modelos Moleculares , Modelos Neurológicos , Estrutura Molecular , Neurônios/fisiologia , Neurotransmissores/química , Técnicas de Patch-Clamp , Piridinas/química , Ratos , Canais de Potássio Shaw/genética , Técnicas de Cultura de TecidosRESUMO
In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.
Assuntos
Epilepsia/genética , Epilepsia/patologia , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Criança , Pré-Escolar , Cromossomos Humanos Par 9 , Epilepsia/diagnóstico , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Canal de Potássio KCNQ2/genética , Masculino , Mutação , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Patologia Molecular , Canais de Potássio Ativados por Sódio , Proteínas Proto-Oncogênicas c-cbl/genética , Dissomia Uniparental , Adulto JovemRESUMO
Voltage-gated K(+) channels of the Shaw family (also known as the KCNC or Kv3 family) play pivotal roles in mammalian brains, and genetic or pharmacological disruption of their activities in mice results in a spectrum of behavioral defects. We have used the model system of Caenorhabditis elegans to elucidate conserved molecular mechanisms that regulate these channels. We have now found that the C. elegans Shaw channel KHT-1, and its mammalian homologue, murine Kv3.1b, are both modulated by acid phosphatases. Thus, the C. elegans phosphatase ACP-2 is stably associated with KHT-1, while its mammalian homolog, prostatic acid phosphatase (PAP; also known as ACPP-201) stably associates with murine Kv3.1b K(+) channels in vitro and in vivo. In biochemical experiments both phosphatases were able to reverse phosphorylation of their associated channel. The effect of phosphorylation on both channels is to produce a decrease in current amplitude and electrophysiological analyses demonstrated that dephosphorylation reversed the effects of phosphorylation on the magnitude of the macroscopic currents. ACP-2 and KHT-1 were colocalized in the nervous system of C. elegans and, in the mouse nervous system, PAP and Kv3.1b were colocalized in subsets of neurons, including in the brain stem and the ventricular zone. Taken together, this body of evidence suggests that acid phosphatases are general regulatory partners of Shaw-like K(+) channels.
Assuntos
Tronco Encefálico/metabolismo , Evolução Molecular , Neurônios/metabolismo , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo , Animais , Tronco Encefálico/patologia , Caenorhabditis elegans , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologiaRESUMO
Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na(+)-activated K(+) channels (K(Na)), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/metabolismo , Neurônios/fisiologia , Canais de Potássio/metabolismo , Animais , Anisomicina/farmacologia , Aplysia , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Fenômenos Eletrofisiológicos , Imuno-Histoquímica , Imunoprecipitação , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Sódio/farmacologia , Sinapses/fisiologiaRESUMO
Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates synaptic plasticity by repressing translation of specific mRNAs. We found that FMRP binds mRNA encoding the voltage-gated potassium channel Kv3.1b in brainstem synaptosomes. To explore the regulation of Kv3.1b by FMRP, we investigated Kv3.1b immunoreactivity and potassium currents in the auditory brainstem sound localization circuit of male mice. The unique features of this circuit allowed us to control neuronal activity in vivo by exposing animals to high-frequency, amplitude-modulated stimuli, which elicit predictable and stereotyped patterns of input to the anterior ventral cochlear nucleus (AVCN) and medial nucleus of the trapezoid body (MNTB). In wild-type (WT) animals, Kv3.1b is expressed along a tonotopic gradient in the MNTB, with highest levels in neurons at the medial, high-frequency end. At baseline, Fmr1(-/-) mice, which lack FMRP, displayed dramatically flattened tonotopicity in Kv3.1b immunoreactivity and K(+) currents relative to WT controls. Moreover, after 30 min of acoustic stimulation, levels of Kv3.1b immunoreactivity were significantly elevated in both the MNTB and AVCN of WT, but not Fmr1(-/-), mice. These results suggest that FMRP is necessary for maintenance of the gradient in Kv3.1b protein levels across the tonotopic axis of the MNTB, and are consistent with a role for FMRP as a repressor of protein translation. Using numerical simulations, we demonstrate that Kv3.1b tonotopicity may be required for accurate encoding of stimulus features such as modulation rate, and that disruption of this gradient, as occurs in Fmr1(-/-) animals, degrades processing of this information.
Assuntos
Vias Auditivas/fisiologia , Tronco Encefálico/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Canais de Potássio Shaw/metabolismo , Estimulação Acústica , Animais , Western Blotting , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Neurônios , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Localização de SomRESUMO
Potassium channels activated by intracellular Na(+) ions (K(Na)) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode K(Na) channels. We have now found that Slick and Slack subunits coassemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization, and response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of the alternative splice variants of the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric K(Na) channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are coexpressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal K(Na) channels.
Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio/química , Canais de Potássio/fisiologia , Processamento Alternativo/genética , Animais , Linhagem Celular , Feminino , Humanos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Canais de Potássio/biossíntese , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Ratos , Xenopus laevisRESUMO
The rates of activation and unitary properties of Na+-activated K+ (K(Na)) currents have been found to vary substantially in different types of neurones. One class of K(Na) channels is encoded by the Slack gene. We have now determined that alternative RNA splicing gives rise to at least five different transcripts for Slack, which produce Slack channels that differ in their predicted cytoplasmic amino-termini and in their kinetic properties. Two of these, termed Slack-A channels, contain an amino-terminus domain closely resembling that of another class of K(Na) channels encoded by the Slick gene. Neuronal expression of Slack-A channels and of the previously described Slack isoform, now called Slack-B, are driven by independent promoters. Slack-A mRNAs were enriched in the brainstem and olfactory bulb and detected at significant levels in four different brain regions. When expressed in CHO cells, Slack-A channels activate rapidly upon depolarization and, in single channel recordings in Xenopus oocytes, are characterized by multiple subconductance states with only brief transient openings to the fully open state. In contrast, Slack-B channels activate slowly over hundreds of milliseconds, with openings to the fully open state that are approximately 6-fold longer than those for Slack-A channels. In numerical simulations, neurones in which outward currents are dominated by a Slack-A-like conductance adapt very rapidly to repeated or maintained stimulation over a wide range of stimulus strengths. In contrast, Slack-B currents promote rhythmic firing during maintained stimulation, and allow adaptation rate to vary with stimulus strength. Using an antibody that recognizes all amino-termini isoforms of Slack, Slack immunoreactivity is present at locations that have no Slack-B-specific staining, including olfactory bulb glomeruli and the dendrites of hippocampal neurones, suggesting that Slack channels with alternate amino-termini such as Slack-A channels are present at these locations. Our data suggest that alternative promoters of the Slack gene differentially modulate the properties of neurones.
Assuntos
Potenciais de Ação/fisiologia , Adaptação Fisiológica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio , Regiões Promotoras Genéticas , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RatosRESUMO
Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.
Assuntos
Membrana Celular/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Canais de Potássio/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Técnicas Biossensoriais , Bitionol/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Proteína do X Frágil da Deficiência Intelectual/antagonistas & inibidores , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/metabolismo , Mutação , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tiazolidinas/farmacologia , Xenopus laevisRESUMO
Impaired tau catabolism may contribute to tau accumulation and aggregation in Alzheimer's disease and neurofibrillary tangle formation. This study examined the effects of proteasome and calpain inhibition on tau levels and turnover in primary rat hippocampal neurons and differentiated SH-SY5Y human neuroblastoma cells. Administration of proteasome (MG-115, lactacystin) or calpain (MDL28170) inhibitors for up to 24 hours did not alter tau levels in differentiated SH-SY5Y cells or rat hippocampal neurons. Addition of 1 microM and 10 microM MG-115 did not change total tau levels, but did result in increased reactivity of phosphorylation-dependent tau antibodies (PHF-1, CP-13) and decreased Tau-1 immunoreactivity. Administration of cycloheximide to inhibit de novo protein synthesis also did not alter tau levels in the presence or absence of lactacystin. These results demonstrate that although the proteasome and calpain protease systems are capable of degrading tau in cell-free assays, their inhibition does not alter cellular tau levels in primary neurons or differentiated neuroblastoma cells.
Assuntos
Calpaína/metabolismo , Inibição Neural/fisiologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Aminopeptidases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Agregação Celular , Hipocampo/imunologia , Hipocampo/metabolismo , Hipocampo/patologia , Técnicas In Vitro , Neuroblastoma/enzimologia , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Neurônios/imunologia , Ratos , Células Tumorais Cultivadas , Proteínas tau/imunologiaRESUMO
Disruptions in energy metabolism have been suggested to be a prominent feature, perhaps even a fundamental component, of Alzheimer's disease (AD). These abnormalities in cerebral metabolism precede the onset of neurological dysfunction as well as gross neuropathology of AD. These changes may stem from inhibition of mitochondrial enzymes including pyruvate dehydrogenase, cytochrome c oxidase, and alpha-ketoglutarate dehydrogenase. Several lines of evidence also suggest a role for oxidative stress in the neuropathology associated with the disease state. Because mitochondria are the major site of free radical production in cells, they are also a primary target for oxidative damage and subsequent dysfunction. This link between mitochondrial dysfunction and the pathophysiology of AD is supported by several lines of evidence.
Assuntos
Envelhecimento/fisiologia , Doença de Alzheimer , Mitocôndrias/fisiologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Córtex Cerebral/fisiopatologia , Expressão Gênica/fisiologia , Humanos , Fatores de TempoRESUMO
Mitochondria are known to be localized in synaptic and non-synaptic compartments in the brain. Synaptoneurosomes, which contain high numbers of mitochondria, may act as a major contaminant of currently used isolation techniques. Currently, there is no method employed to successfully disrupt synaptoneurosomes and isolate both synaptic and non-synaptic mitochondria without structural or functional damage. A novel method is reported here for disruption of synaptoneurosomes and isolation of total brain mitochondria from synaptic and non-synaptic sources using a nitrogen decompression technique. Nitrogen gas was dissolved into crude mitochondrial preparations and maintained under constant, moderate pressure. After a short incubation, the pressure was released causing the nitrogen to come out of solution as growing bubbles, which ruptures cellular and synaptoneurosomal membranes. Mitochondria isolated using this rapid technique were bioenergetically competent and exhibited functional characteristics comparable to mitochondria isolated using traditional techniques. This nitrogen decompression technique will allow for further characterization of synaptic pools of mitochondria, which are almost exclusively neuronal in origin.
Assuntos
Encéfalo/citologia , Técnicas Citológicas/métodos , Mitocôndrias , Nitrogênio/farmacologia , Sinaptossomos/efeitos dos fármacos , Animais , Western Blotting/métodos , Digitonina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Masculino , Microscopia Eletrônica/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Consumo de Oxigênio , Porinas , Ratos , Frações Subcelulares , Canais de Ânion Dependentes de VoltagemRESUMO
Neuronal dendrites are structurally and functionally dynamic in response to changes in afferent activity. The fragile X mental retardation protein (FMRP) is an mRNA binding protein that regulates activity-dependent protein synthesis and morphological dynamics of dendrites. Loss and abnormal expression of FMRP occur in fragile X syndrome (FXS) and some forms of autism spectrum disorders. To provide further understanding of how FMRP signaling regulates dendritic dynamics, we examined dendritic expression and localization of FMRP in the reptilian and avian nucleus laminaris (NL) and its mammalian analogue, the medial superior olive (MSO), in rodents and humans. NL/MSO neurons are specialized for temporal processing of low-frequency sounds for binaural hearing, which is impaired in FXS. Protein BLAST analyses first demonstrate that the FMRP amino acid sequences in the alligator and chicken are highly similar to human FMRP with identical mRNA-binding and phosphorylation sites, suggesting that FMRP functions similarly across vertebrates. Immunocytochemistry further reveals that NL/MSO neurons have very high levels of dendritic FMRP in low-frequency hearing vertebrates including alligator, chicken, gerbil, and human. Remarkably, dendritic FMRP in NL/MSO neurons often accumulates at branch points and enlarged distal tips, loci known to be critical for branch-specific dendritic arbor dynamics. These observations support an important role for FMRP in regulating dendritic properties of binaural neurons that are essential for low-frequency sound localization and auditory scene segregation, and support the relevance of studying this regulation in nonhuman vertebrates that use low frequencies in order to further understand human auditory processing disorders.
Assuntos
Jacarés e Crocodilos/metabolismo , Tronco Encefálico/metabolismo , Galinhas/metabolismo , Dendritos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Gerbillinae/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Humanos , Masculino , Pessoa de Meia-Idade , Ratos Sprague-Dawley/metabolismo , Proteínas de Répteis/genética , Proteínas de Répteis/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Complexo Olivar Superior/metabolismoRESUMO
Malignant migrating partial seizures of infancy (MMPSI) is a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with developmental delay. We performed exome sequencing in three probands with MMPSI and identified de novo gain-of-function mutations affecting the C-terminal domain of the KCNT1 potassium channel. We sequenced KCNT1 in 9 additional individuals with MMPSI and identified mutations in 4 of them, in total identifying mutations in 6 out of 12 unrelated affected individuals. Functional studies showed that the mutations led to constitutive activation of the channel, mimicking the effects of phosphorylation of the C-terminal domain by protein kinase C. In addition to regulating ion flux, KCNT1 has a non-conducting function, as its C terminus interacts with cytoplasmic proteins involved in developmental signaling pathways. These results provide a focus for future diagnostic approaches and research for this devastating condition.
Assuntos
Epilepsias Parciais/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Neurônios , Animais , Células Cultivadas , Eletroencefalografia , Epilepsias Parciais/fisiopatologia , Exoma , Humanos , Lactente , Recém-Nascido , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Camundongos , Mutação , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Ratos , Transdução de Sinais , XenopusRESUMO
For accurate processing of auditory information, neurons in auditory brainstem nuclei have to fire at high rates with high temporal accuracy. These two requirements can only be fulfilled when the intrinsic electrical properties of these neurons are matched to the pattern of incoming synaptic stimulation. This review article focuses on three families of potassium channels that are critical to shaping the firing pattern and accuracy of neurons. Changes in the auditory environment can trigger very rapid changes in the phosphorylation state of potassium channels in auditory brainstem nuclei. Longer lasting changes in the auditory environment produce changes in the rates of translation and transcription of genes encoding these channels. A key protein that plays a role in setting the overall sensitivity of the auditory system to sound stimuli is FMRP (Fragile X Mental Retardation Protein), which binds channels directly and also regulates the translation of mRNAs for the channels.
Assuntos
Vias Auditivas/fisiologia , Tronco Encefálico/metabolismo , Canais de Potássio/metabolismo , Potenciais de Ação , Animais , Tronco Encefálico/fisiologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Fosforilação , Reprodutibilidade dos Testes , Canais de Potássio Shaw/metabolismo , Transdução de Sinais , Sódio/farmacologia , Transcrição GênicaRESUMO
In humans, the absence of Fragile X mental retardation protein (FMRP), an RNA-binding protein, results in Fragile X syndrome, the most common inherited form of intellectual disability. Using biochemical and electrophysiological studies, we found that FMRP binds to the C terminus of the Slack sodium-activated potassium channel to activate the channel in mice. Our findings suggest that Slack activity provides a link between patterns of neuronal firing and changes in protein translation.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/fisiologia , Ativação do Canal Iônico/fisiologia , Canais de Potássio/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Proteínas do Tecido Nervoso , Canais de Potássio Ativados por SódioRESUMO
Mitochondria in nerve terminals are subjected to extensive Ca2+ fluxes and high energy demands, but the extent to which the synaptic mitochondria buffer Ca2+ is unclear. In this study, we identified a difference in the Ca2+ clearance ability of nonsynaptic versus synaptic mitochondrial populations enriched from rat cerebral cortex. Mitochondria were isolated using Percoll discontinuous gradients in combination with high pressure nitrogen cell disruption. Mitochondria in the nonsynaptic fraction originate from neurons and other cell types including glia, whereas mitochondria enriched from a synaptosomal fraction are predominantly neuronal and presynaptic in origin. There were no differences in respiration or initial Ca2+ loads between nonsynaptic and synaptic mitochondrial populations. Following both bolus and infusion Ca2+ addition, nonsynaptic mitochondria were able to accumulate significantly more exogenously added Ca2+ than the synaptic mitochondria before undergoing mitochondrial permeability transition, observed as a loss in mitochondrial membrane potential and decreased Ca2+ uptake. The limited ability of synaptic mitochondria to accumulate Ca2+ could result from several factors including a primary function of ATP production to support the high energy demand of presynaptic terminals, their relative isolation in comparison with the threads or clusters of mitochondria found in the soma of neurons and glia, or the older age and increased exposure to oxidative damage of synaptic versus nonsynaptic mitochondria. By more readily undergoing permeability transition, synaptic mitochondria may initiate neuron death in response to insults that elevate synaptic levels of intracellular Ca2+, consistent with the early degeneration of distal axon segments in neurodegenerative disorders.
Assuntos
Cálcio/metabolismo , Mitocôndrias/metabolismo , Sinapses/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular , Córtex Cerebral/metabolismo , Masculino , Potenciais da Membrana , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
An age-related Ca(2+) dysregulation and increased production of reactive oxygen species (ROS) may contribute to late-onset neurodegenerative disorders. These alterations are often attributed to impaired mitochondrial function yet few studies have directly examined mitochondria isolated from various regions of the aged brain. The purpose of this study was to examine Ca(2+)-buffering and ROS production in mitochondria isolated from Fischer 344 rats ranging in age from 4 to 25 months. Mitchondria isolated from the cortex of the 25 month rat brain exhibited greater rates of ROS production and mitochondrial swelling in response to increasing Ca(2+) loads as compared to mitochondria isolated from younger (4, 13 month) animals. The increased swelling is indicative of opening of the mitochondrial permeability transition pore indicating impaired Ca(2+) buffering/cycling in aged animals. These age-related differences were not observed in mitochondria isolated from cerebellum. Together, these results demonstrate region specific, age-related, alterations in mitochondrial responses to Ca(2+).