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1.
Cell ; 165(2): 434-448, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26997484

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/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(12): e2216440120, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36930599

RESUMO

Potassium channels in auditory neurons are rapidly modified by changes in the auditory environment. In response to elevated auditory stimulation, short-term mechanisms such as protein phosphorylation and longer-term mechanisms such as accelerated channel synthesis increase the amplitude of currents that promote high-frequency firing. It has been suggested that this allows neurons to fire at high rates in response to high sound levels. We have carried out simple simulations of the response to postsynaptic neurons to patterns of neurotransmitter release triggered by auditory stimuli. These demonstrate that the amplitudes of potassium currents required for optimal encoding of a low-amplitude auditory signal differ from those for louder sounds. Specifically, the cross-correlation of the output of a neuron with an auditory stimulus is improved by increasing potassium currents as sound amplitude increases. Temporal fidelity for low-frequency stimuli is improved by increasing potassium currents that activate at negative potentials, while that for high-frequency stimuli requires increases in currents that activate at positive membrane potentials. These effects are independent of the firing rate. Moreover, levels of potassium currents that maximize the fidelity of the output of an ensemble of neurons differ from those that maximize fidelity for a single neuron. This suggests that the modulatory mechanisms must coordinate channel activity in groups of neurons or an entire nucleus. The simulations provide an explanation for the modulation of the intrinsic excitability of auditory brainstem neurons by changes in environmental sound levels, and the results may extend to information processing in other neural systems.


Assuntos
Canais de Potássio , Potássio , Potenciais de Ação/fisiologia , Potenciais da Membrana , Canais de Potássio/metabolismo , Fosforilação , Potássio/metabolismo , Vias Auditivas/fisiologia
3.
Physiol Rev ; 97(4): 1431-1468, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28904001

RESUMO

The intrinsic electrical characteristics of different types of neurons are shaped by the K+ channels they express. From among the more than 70 different K+ channel genes expressed in neurons, Kv3 family voltage-dependent K+ channels are uniquely associated with the ability of certain neurons to fire action potentials and to release neurotransmitter at high rates of up to 1,000 Hz. In general, the four Kv3 channels Kv3.1-Kv3.4 share the property of activating and deactivating rapidly at potentials more positive than other channels. Each Kv3 channel gene can generate multiple protein isoforms, which contribute to the high-frequency firing of neurons such as auditory brain stem neurons, fast-spiking GABAergic interneurons, and Purkinje cells of the cerebellum, and to regulation of neurotransmitter release at the terminals of many neurons. The different Kv3 channels have unique expression patterns and biophysical properties and are regulated in different ways by protein kinases. In this review, we cover the function, localization, and modulation of Kv3 channels and describe how levels and properties of the channels are altered by changes in ongoing neuronal activity. We also cover how the protein-protein interaction of these channels with other proteins affects neuronal functions, and how mutations or abnormal regulation of Kv3 channels are associated with neurological disorders such as ataxias, epilepsies, schizophrenia, and Alzheimer's disease.


Assuntos
Neurônios/metabolismo , Neurotransmissores/metabolismo , Canais de Potássio Shaw/metabolismo , Sequência de Aminoácidos , Animais , Orientação de Axônios , Humanos , Dados de Sequência Molecular , Doenças do Sistema Nervoso/metabolismo , Fosfotransferases/metabolismo , Canais de Potássio Shaw/antagonistas & inibidores , Transdução de Sinais
4.
Proc Biol Sci ; 289(1980): 20220878, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35946148

RESUMO

Life underground often leads to animals having specialized auditory systems to accommodate the constraints of acoustic transmission in tunnels. Despite living underground, naked mole-rats use a highly vocal communication system, implying that they rely on central auditory processing. However, little is known about these animals' central auditory system, and whether it follows a similar developmental time course as other rodents. Naked mole-rats show slowed development in the hippocampus suggesting they have altered brain development compared to other rodents. Here, we measured morphological characteristics and voltage-gated potassium channel Kv3.3 expression and protein levels at different key developmental time points (postnatal days 9, 14, 21 and adulthood) to determine whether the auditory brainstem (lateral superior olive and medial nucleus of the trapezoid body) develops similarly to two common auditory rodent model species: gerbils and mice. Additionally, we measured the hearing onset of naked mole-rats using auditory brainstem response recordings at the same developmental timepoints. In contrast with other work in naked mole-rats showing that they are highly divergent in many aspects of their physiology, we show that naked mole-rats have a similar hearing onset, between postnatal day (P) 9 and P14, to many other rodents. On the other hand, we show some developmental differences, such as a unique morphology and Kv3.3 protein levels in the brainstem.


Assuntos
Tronco Encefálico , Ratos-Toupeira , Animais , Percepção Auditiva/fisiologia , Tronco Encefálico/anatomia & histologia , Gerbillinae , Hipocampo , Camundongos , Ratos-Toupeira/fisiologia
5.
FASEB J ; 35(12): e22053, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34820911

RESUMO

Mutations in KCNC3, the gene that encodes the Kv3.3 voltage dependent potassium channel, cause Spinocerebellar Ataxia type 13 (SCA13), a disease associated with disrupted motor behaviors, progressive cerebellar degeneration, and abnormal auditory processing. The Kv3.3 channel directly binds Hax-1, a cell survival protein. A disease-causing mutation, Kv3.3-G592R, causes overstimulation of Tank Binding Kinase 1 (Tbk1) in the cerebellum, resulting in the degradation of Hax-1 by promoting its trafficking into multivesicular bodies and then to lysosomes. We have now tested the effects of antisense oligonucleotides (ASOs) directed against the Kv3.3 channel on both wild type mice and those bearing the Kv3.3-G592R-encoding mutation. Intracerebroventricular infusion of the Kcnc3-specific ASO suppressed both mRNA and protein levels of the Kv3.3 channel. In wild-type animals, this produced no change in levels of activated Tbk1, Hax-1 or Cd63, a tetraspanin marker for late endosomes/multivesicular bodies. In contrast, in mice homozygous for the Kv3.3-G592R-encoding mutation, the same ASO reduced Tbk1 activation and levels of Cd63, while restoring the expression of Hax-1 in the cerebellum. The motor behavior of the mice was tested using a rotarod assay. Surprisingly, the active ASO had no effects on the motor behavior of wild type mice but restored the behavior of the mutant mice to those of age-matched wild type animals. Our findings indicate that, in mature intact animals, suppression of Kv3.3 expression can reverse the deleterious effects of a SCA13 mutation while having little effect on wild type animals. Thus, targeting Kv3.3 expression may prove a viable therapeutic approach for SCA13.


Assuntos
Transtornos Motores/prevenção & controle , Mutação , Oligonucleotídeos Antissenso/administração & dosagem , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Potássio Shaw/antagonistas & inibidores , Ataxias Espinocerebelares/complicações , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transtornos Motores/etiologia , Transtornos Motores/metabolismo , Transtornos Motores/patologia , Proteínas Serina-Treonina Quinases/genética , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo
6.
Platelets ; 33(3): 451-461, 2022 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-34348571

RESUMO

Kv1.3 is a voltage-gated K+-selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood. Here we show that Kv1.3-deficient platelets display enhanced ADP-evoked platelet aggregation and secretion, and an increased surface expression of platelet integrin αIIb. In contrast, platelet adhesion and thrombus formation in vitro under arterial shear conditions on surfaces coated with collagen were reduced for samples from Kv1.3-/- compared to wild type mice. Use of collagen-mimetic peptides revealed a specific defect in the engagement with α2ß1. Kv1.3-/- platelets developed significantly fewer, and shorter, filopodia than wild type platelets during adhesion to collagen fibrils. Kv1.3-/- mice displayed no significant difference in thrombus formation within cremaster muscle arterioles using a laser-induced injury model, thus other pro-thrombotic pathways compensate in vivo for the adhesion defect observed in vitro. This may include the increased platelet counts of Kv1.3-/- mice, due in part to a prolonged lifespan. The ability of Kv1.3 to modulate integrin-dependent platelet adhesion has important implications for understanding its contribution to normal physiological platelet function in addition to its reported roles in auto-immune diseases and thromboinflammatory models of stroke.


Assuntos
Plaquetas/metabolismo , Colágeno/metabolismo , Integrina alfa2beta1/metabolismo , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Humanos
7.
J Neurophysiol ; 126(2): 532-539, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34232791

RESUMO

Channelopathies caused by mutations in genes encoding ion channels generally produce a clear change in channel function. Accordingly, mutations in KCNC1, which encodes the voltage-dependent Kv3.1 potassium channel, result in progressive myoclonus epilepsy as well as other developmental and epileptic encephalopathies, and these have been shown to reduce or fully abolish current amplitude. One exception to this is the mutation A513V Kv3.1b, located in the cytoplasmic C-terminal domain of the channel protein. This de novo variant was detected in a patient with epilepsy of infancy with focal migrating seizures (EIFMS), but no difference could be detected between A513V Kv3.1 current and that of wild-type Kv3.1. Using both biochemical and electrophysiological approaches, we have now confirmed that this variant produces functional channels but find that the A513V mutation renders the channel completely insensitive to regulation by phosphorylation at S503, a nearby regulatory site in the C-terminus. In this respect, the mutation resembles those in another channel, KCNT1, which are the major cause of EIFMS. Because the amplitude of Kv3.1 current is constantly adjusted by phosphorylation in vivo, our findings suggest that loss of such regulation contributes to EIFMS phenotype and emphasize the role of channel modulation for normal neuronal function.NEW & NOTEWORTHY Ion channel mutations that cause serious human diseases generally alter the biophysical properties or expression of the channel. We describe a de novo mutation in the Kv3.1 potassium channel that causes severe intellectual disability with early-onset epilepsy. The properties of this channel appear identical to those of wild-type channels, but the mutation prevents phosphorylation of the channel by protein kinase C. Our findings emphasize the role of channel modulation in normal brain function.


Assuntos
Epilepsia/genética , Mutação , Canais de Potássio Shaw/metabolismo , Sialiltransferases/deficiência , Animais , Células CHO , Cricetinae , Cricetulus , Epilepsia/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Canais de Potássio Shaw/química , Canais de Potássio Shaw/genética , Sialiltransferases/genética , Sialiltransferases/metabolismo
8.
FASEB J ; 34(3): 3501-3518, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32039504

RESUMO

Autism spectrum disorders (ASD) are strongly associated with auditory hypersensitivity or hyperacusis (difficulty tolerating sounds). Fragile X syndrome (FXS), the most common monogenetic cause of ASD, has emerged as a powerful gateway for exploring underlying mechanisms of hyperacusis and auditory dysfunction in ASD. This review discusses examples of disruption of the auditory pathways in FXS at molecular, synaptic, and circuit levels in animal models as well as in FXS individuals. These examples highlight the involvement of multiple mechanisms, from aberrant synaptic development and ion channel deregulation of auditory brainstem circuits, to impaired neuronal plasticity and network hyperexcitability in the auditory cortex. Though a relatively new area of research, recent discoveries have increased interest in auditory dysfunction and mechanisms underlying hyperacusis in this disorder. This rapidly growing body of data has yielded novel research directions addressing critical questions regarding the timing and possible outcomes of human therapies for auditory dysfunction in ASD.


Assuntos
Transtorno do Espectro Autista/fisiopatologia , Síndrome do Cromossomo X Frágil/fisiopatologia , Animais , Percepção Auditiva/fisiologia , Transtorno do Espectro Autista/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Modelos Biológicos
9.
J Neurosci ; 39(24): 4797-4813, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-30936239

RESUMO

Fragile X syndrome (FXS) is characterized by hypersensitivity to sensory stimuli, including environmental sounds. We compared the auditory brainstem response (ABR) recorded in vivo in mice lacking the gene (Fmr1-/y ) for fragile X mental retardation protein (FMRP) with that in wild-type animals. We found that ABR wave I, which represents input from the auditory nerve, is reduced in Fmr1-/y animals, but only at high sound levels. In contrast, wave IV, which represents the activity of auditory brainstem nuclei is enhanced at all sound levels, suggesting that loss of FMRP alters the central processing of auditory signals. Current-clamp recordings of neurons in the medial nucleus of the trapezoid body in the auditory brainstem revealed that, in contrast to neurons from wild-type animals, sustained depolarization triggers repetitive firing rather than a single action potential. In voltage-clamp recordings, K+ currents that activate at positive potentials ("high-threshold" K+ currents), which are required for high-frequency firing and are carried primarily by Kv3.1 channels, are elevated in Fmr1-/y mice, while K+ currents that activate near the resting potential and inhibit repetitive firing are reduced. We therefore tested the effects of AUT2 [((4-({5-[(4R)-4-ethyl-2,5-dioxo-1-imidazolidinyl]-2-pyridinyl}oxy)-2-(1-methylethyl) benzonitrile], a compound that modulates Kv3.1 channels. AUT2 reduced the high-threshold K+ current and increased the low-threshold K+ currents in neurons from Fmr1-/y animals by shifting the activation of the high-threshold current to more negative potentials. This reduced the firing rate and, in vivo, restored wave IV of the ABR. Our results from animals of both sexes suggest that the modulation of the Kv3.1 channel may have potential for the treatment of sensory hypersensitivity in patients with FXS.SIGNIFICANCE STATEMENT mRNA encoding the Kv3.1 potassium channel was one of the first described targets of the fragile X mental retardation protein (FMRP). Fragile X syndrome is caused by loss of FMRP and, in humans and mice, causes hypersensitivity to auditory stimuli. We found that components of the auditory brain response (ABR) corresponding to auditory brainstem activity are enhanced in mice lacking FMRP. This is accompanied by hyperexcitability and altered potassium currents in auditory brainstem neurons. Treatment with a drug that alters the voltage dependence of Kv3.1 channels normalizes the imbalance of potassium currents, as well as ABR responses in vivo, suggesting that such compounds may be effective in treating some symptoms of fragile X syndrome.


Assuntos
Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/metabolismo , Canais de Potássio Shaw/metabolismo , Animais , Vias Auditivas , Percepção Auditiva , Tronco Encefálico/efeitos dos fármacos , Núcleo Coclear/fisiologia , Fenômenos Eletrofisiológicos , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Feminino , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Síndrome do Cromossomo X Frágil/genética , Hidantoínas/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Piridinas/farmacologia
10.
J Neurosci ; 39(37): 7438-7449, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31350261

RESUMO

Mutations in the KCNT1 (Slack, KNa1.1) sodium-activated potassium channel produce severe epileptic encephalopathies. Expression in heterologous systems has shown that the disease-causing mutations give rise to channels that have increased current amplitude. It is not known, however, whether such gain of function occurs in human neurons, nor whether such increased KNa current is expected to suppress or increase the excitability of cortical neurons. Using genetically engineered human induced pluripotent stem cell (iPSC)-derived neurons, we have now found that sodium-dependent potassium currents are increased several-fold in neurons bearing a homozygous P924L mutation. In current-clamp recordings, the increased KNa current in neurons with the P924L mutation acts to shorten the duration of action potentials and to increase the amplitude of the afterhyperpolarization that follows each action potential. Strikingly, the number of action potentials that were evoked by depolarizing currents as well as maximal firing rates were increased in neurons expressing the mutant channel. In networks of spontaneously active neurons, the mean firing rate, the occurrence of rapid bursts of action potentials, and the intensity of firing during the burst were all increased in neurons with the P924L Slack mutation. The feasibility of an increased KNa current to increase firing rates independent of any compensatory changes was validated by numerical simulations. Our findings indicate that gain-of-function in Slack KNa channels causes hyperexcitability in both isolated neurons and in neural networks and occurs by a cell-autonomous mechanism that does not require network interactions.SIGNIFICANCE STATEMENTKCNT1 mutations lead to severe epileptic encephalopathies for which there are no effective treatments. This study is the first demonstration that a KCNT1 mutation increases the Slack current in neurons. It also provides the first explanation for how this increased potassium current induces hyperexcitability, which could be the underlining factor causing seizures.


Assuntos
Epilepsia/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação/fisiologia , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Canais de Potássio Ativados por Sódio/genética , Potenciais de Ação/fisiologia , Diferenciação Celular/fisiologia , Epilepsia/fisiopatologia , Células HEK293 , Humanos
11.
Pharmacol Rev ; 69(1): 1-11, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28267675

RESUMO

A subset of potassium channels is regulated primarily by changes in the cytoplasmic concentration of ions, including calcium, sodium, chloride, and protons. The eight members of this subfamily were originally all designated as calcium-activated channels. More recent studies have clarified the gating mechanisms for these channels and have documented that not all members are sensitive to calcium. This article describes the molecular relationships between these channels and provides an introduction to their functional properties. It also introduces a new nomenclature that differentiates between calcium- and sodium-activated potassium channels.


Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Ativação do Canal Iônico , Canais de Potássio Cálcio-Ativados/classificação , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Potássio/classificação , Canais de Potássio/metabolismo , Sódio/metabolismo , Terminologia como Assunto , Animais , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/classificação , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/classificação , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Espermatozoides/metabolismo
12.
J Neurosci ; 37(8): 2258-2265, 2017 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-28119399

RESUMO

Mutations that alter levels of Slack (KCNT1) Na+-activated K+ current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica, a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na+ from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na+-activated K+ channels in neurons.SIGNIFICANCE STATEMENT Slack Na+-activated K+ channels (KCNT1, KNa1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal development and function. We find that injection of oligomers of mutant superoxide dismutase 1 (SOD1) into the cytoplasm of invertebrate neurons rapidly suppresses these Na+-activated K+ currents and that this effect is mediated by a MAP kinase cascade, including ASK1 and c-Jun N-terminal kinase. Because amyotrophic lateral sclerosis is a fatal adult-onset neurodegenerative disease produced by mutations in SOD1 that cause the enzyme to form toxic oligomers, our findings suggest that suppression of Slack channels may be an early step in the progression of the disease.


Assuntos
Potenciais da Membrana/genética , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Canais de Potássio/metabolismo , Superóxido Dismutase-1/genética , Animais , Aplysia/citologia , Biofísica , Células Cultivadas , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Gânglios dos Invertebrados/citologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Microinjeções , Morfolinos/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio Ativados por Sódio , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Sódio/farmacologia , Superóxido Dismutase-1/química
13.
Cerebellum ; 17(5): 692-697, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29949095

RESUMO

Mutations in the potassium channel gene KCNC3 (Kv3.3) cause the autosomal dominant neurological disease, spinocerebellar ataxia 13 (SCA13). In this study, we expand the genotype-phenotype repertoire of SCA13 by describing the novel KCNC3 deletion p.Pro583_Pro585del highlighting the allelic heterogeneity observed in SCA13 patients. We characterize adult-onset, progressive clinical symptoms of two afflicted kindred and introduce the symptom of profound spasticity not previously associated with the SCA13 phenotype. We also present molecular and electrophysiological characterizations of the mutant protein in mammalian cell culture. Mechanistically, the p.Pro583_Pro585del protein showed normal membrane trafficking with an altered electrophysiological profile, including slower inactivation and decreased sensitivity to the inactivation-accelerating effects of the actin depolymerizer latrunculin B. Taken together, our results highlight the clinical importance of the intracellular C-terminal portion of Kv3.3 and its association with ion channel function.


Assuntos
Espasticidade Muscular/genética , Espasticidade Muscular/fisiopatologia , Deleção de Sequência , Canais de Potássio Shaw/genética , Ataxias Espinocerebelares/congênito , Adulto , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células CHO , Cricetulus , Feminino , Humanos , Masculino , Toxinas Marinhas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Espasticidade Muscular/diagnóstico por imagem , Fenótipo , Ataxias Espinocerebelares/diagnóstico por imagem , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia , Tiazolidinas/farmacologia
14.
J Physiol ; 594(16): 4677-84, 2016 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-26442672

RESUMO

The voltage-dependent potassium channel subunit Kv3.3 is expressed at high levels in cerebellar Purkinje cells, in auditory brainstem nuclei and in many other neurons capable of firing at high rates. In the cerebellum, it helps to shape the very characteristic complex spike of Purkinje cells. Kv3.3 differs from other closely related channels in that human mutations in the gene encoding Kv3.3 (KCNC3) result in a unique neurodegenerative disease termed spinocerebellar ataxia type 13 (SCA13). This primarily affects the cerebellum, but also results in extracerebellar symptoms. Different mutations produce either early onset SCA13, associated with delayed motor and impaired cognitive skill acquisition, or late onset SCA13, which typically produces cerebellar degeneration in middle age. This review covers the localization and physiological function of Kv3.3 in the central nervous system and how the normal function of the channel is altered by the disease-causing mutations. It also describes experimental approaches that are being used to understand how Kv3.3 mutations are linked to neuronal survival, and to develop strategies for treatment.


Assuntos
Canais de Potássio Shaw/fisiologia , Ataxias Espinocerebelares/congênito , Animais , Humanos , Canais de Potássio Shaw/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/fisiopatologia
15.
J Neurophysiol ; 116(1): 106-21, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27052580

RESUMO

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 Tecidos
16.
Hum Mol Genet ; 23(12): 3200-11, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24463883

RESUMO

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 Jovem
17.
Learn Mem ; 22(7): 323-35, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26077685

RESUMO

Kcnt1 encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels.


Assuntos
Adaptação Psicológica/fisiologia , Cognição/fisiologia , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Reversão de Aprendizagem/fisiologia , Animais , Cerebelo/metabolismo , Comportamento Exploratório/fisiologia , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Motivação/fisiologia , Atividade Motora/fisiologia , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Canais de Potássio Ativados por Sódio
18.
J Neurosci ; 34(46): 15159-69, 2014 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-25392484

RESUMO

Voltage- and ligand-gated ion channels form the molecular basis of cellular excitability. With >400 members and accounting for ∼1.5% of the human genome, ion channels are some of the most well studied of all proteins in heterologous expression systems. Yet, ion channels often exhibit unexpected properties in vivo because of their interaction with a variety of signaling/scaffolding proteins. Such interactions can influence the function and localization of ion channels, as well as their coupling to intracellular second messengers and pathways, thus increasing the signaling potential of these ion channels in neurons. Moreover, functions have been ascribed to ion channels that are largely independent of their ion-conducting roles. Molecular and functional dissection of the ion channel proteome/interactome has yielded new insights into the composition of ion channel complexes and how their dysregulation leads to human disease.


Assuntos
Canais Iônicos/fisiologia , Proteômica , Transdução de Sinais/fisiologia , Animais , Adesão Celular/fisiologia , Humanos , Ativação do Canal Iônico/fisiologia , Neurônios/fisiologia , Subunidades Proteicas/fisiologia
19.
FASEB J ; 27(4): 1381-93, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23233530

RESUMO

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/fisiologia
20.
Cell Rep ; 43(3): 113904, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38457342

RESUMO

The KCNT1 gene encodes the sodium-activated potassium channel Slack (KCNT1, KNa1.1), a regulator of neuronal excitability. Gain-of-function mutations in humans cause cortical network hyperexcitability, seizures, and severe intellectual disability. Using a mouse model expressing the Slack-R455H mutation, we find that Na+-dependent K+ (KNa) and voltage-dependent sodium (NaV) currents are increased in both excitatory and inhibitory cortical neurons. These increased currents, however, enhance the firing of excitability neurons but suppress that of inhibitory neurons. We further show that the expression of NaV channel subunits, particularly that of NaV1.6, is upregulated and that the length of the axon initial segment and of axonal NaV immunostaining is increased in both neuron types. Our study on the coordinate regulation of KNa currents and the expression of NaV channels may provide an avenue for understanding and treating epilepsies and other neurological disorders.


Assuntos
Epilepsia , Canais de Potássio , Humanos , Axônios/metabolismo , Epilepsia/genética , Mutação/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Animais , Camundongos
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