Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
1.
Elife ; 82019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31774395

RESUMO

In area CA1 of the hippocampus, the selection of place cells to represent a new environment is biased towards neurons with higher excitability. However, different environments are represented by orthogonal cell ensembles, suggesting that regulatory mechanisms exist. Activity-dependent plasticity of intrinsic excitability, as observed in vitro, is an attractive candidate. Here, using whole-cell patch-clamp recordings of CA1 pyramidal neurons in anesthetized rats, we have examined how inducing theta-bursts of action potentials affects their intrinsic excitability over time. We observed a long-lasting, homeostatic depression of intrinsic excitability which commenced within minutes, and, in contrast to in vitro observations, was not mediated by dendritic Ih. Instead, it was attenuated by the Kv1.1 channel blocker dendrotoxin K, suggesting an axonal origin. Analysis of place cells' out-of-field firing in mice navigating in virtual reality further revealed an experience-dependent reduction consistent with decreased excitability. We propose that this mechanism could reduce memory interference.


Assuntos
Região CA1 Hipocampal/fisiologia , Homeostase/fisiologia , Canal de Potássio Kv1.1/metabolismo , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Potenciais de Ação/fisiologia , Animais , Axônios/metabolismo , Quelantes de Cálcio/farmacologia , Dendritos/fisiologia , Eletrofisiologia , Hipocampo/fisiologia , Canal de Potássio Kv1.1/efeitos dos fármacos , Masculino , Camundongos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Peptídeos/antagonistas & inibidores , Ratos , Ratos Wistar
2.
Curr Pharm Des ; 20(2): 189-200, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23701546

RESUMO

Plasma membrane (PM) and mitochondrial (mt) ion channels - particularly potassium channels - became oncological targets soon after the discovery that they are involved both in the regulation of proliferation and apoptosis. Some members of the Kv Shaker family, namely Kv1.1, Kv1.3, Kv1.5 and Kv11.1 (Herg), and the intermediate-conductance calcium-activated potassium KCa3.1 (IK) channels have been shown to contribute to apoptosis in various cell lines. Kv1.3, Kv1.5 and IK are located in the plasma membrane but also in the mitochondrial inner membrane, where they participate in apoptotic signalling. Interestingly, an altered protein expression of some of the channels mentioned above has been reported in neoplastic cell lines/tissues, but a systematic quantification addressing the protein expression of the above potassium channels in tumor cell lines of different origin has not been carried out yet. In the present study we investigated whether expression of specific potassium channels, at the mRNA and protein level, can be correlated with cell sensitivity to various apoptotic stimuli, including chemotherapeutic drugs, in a panel of cancer cell lines. The results show correlation between the protein expression of the Kv1.1 and Kv1.3 channels and susceptibility to death upon treatment with staurosporine, C2-ceramide and cisplatin. Furthermore, we investigated the correlation between Kv channel expression and sensitivity to three distinct membrane-permeant Kv1.3 inhibitors, since these drugs have recently been shown to be able to induce apoptosis and also reduce tumor volume in an in vivo model. Higher protein expression of Kv1.3 significantly correlated with lower cell survival upon treatment with clofazimine, one of the Kv1.3 inhibitors. These results suggest that expression of Kv1.1 and Kv1.3 sensitizes tumour cells of various origins to cytotoxins. Data reported in this work regarding potassium channel protein expression in different cancer cell lines may be exploited for pharmacological manipulation aiming to affect proliferation/apoptosis of cancer cells.


Assuntos
Antineoplásicos/farmacologia , Canal de Potássio Kv1.1/efeitos dos fármacos , Canal de Potássio Kv1.3/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/metabolismo , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro/metabolismo , Esfingosina/análogos & derivados , Esfingosina/farmacologia , Estaurosporina/farmacologia , Carga Tumoral/efeitos dos fármacos
3.
Brain Res ; 1435: 154-66, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-22206926

RESUMO

Mutations in the KCNA1 gene, which encodes for the α subunit of the voltage-gated potassium channel Kv1.1, cause episodic ataxia type 1 (EA1). EA1 is a dominant human neurological disorder characterized by variable phenotypes of brief episodes of ataxia, myokymia, neuromyotonia, and associated epilepsy. Animal models for EA1 include Kcna1-deficient mice, which recessively display severe seizures and die prematurely, and V408A-knock-in mice, which dominantly exhibit stress-induced loss of motor coordination. In the present study, we have identified an N-ethyl-N-nitrosourea-mutagenized rat, named autosomal dominant myokymia and seizures (ADMS), with a missense mutation (S309T) in the voltage-sensor domain, S4, of the Kcna1 gene. ADMS rats dominantly exhibited myokymia, neuromyotonia and generalized tonic-clonic seizures. They also showed cold stress-induced tremor, neuromyotonia, and motor incoordination. Expression studies of homomeric and heteromeric Kv1.1 channels in HEK cells and Xenopus oocytes, showed that, although S309T channels are transferred to the cell membrane surface, they remained non-functional in terms of their biophysical properties, suggesting a dominant-negative effect of the S309T mutation on potassium channel function. ADMS rats provide a new model, distinct from previously reported mouse models, for studying the diverse functions of Kv1.1 in vivo, as well as for understanding the pathology of EA1.


Assuntos
Epilepsia/genética , Síndrome de Isaacs/genética , Canal de Potássio Kv1.1/genética , Mutagênese/efeitos dos fármacos , Mutação de Sentido Incorreto/efeitos dos fármacos , Mioquimia/genética , Animais , Antieméticos/uso terapêutico , Fenômenos Biofísicos/genética , Biotinilação , Peso Corporal/efeitos dos fármacos , Carbamazepina/uso terapêutico , Células Cultivadas , Mapeamento Cromossômico , Análise Mutacional de DNA , Modelos Animais de Doenças , Estimulação Elétrica , Eletroencefalografia , Eletromiografia , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Epilepsia/mortalidade , Etilnitrosoureia/toxicidade , Técnicas de Transferência de Genes , Humanos , Síndrome de Isaacs/induzido quimicamente , Síndrome de Isaacs/mortalidade , Canal de Potássio Kv1.1/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Modelos Moleculares , Mutagênicos/toxicidade , Mutação de Sentido Incorreto/genética , Mioquimia/induzido quimicamente , Mioquimia/mortalidade , Oócitos , Técnicas de Patch-Clamp , Transporte Proteico/genética , Desempenho Psicomotor/fisiologia , Ratos , Ratos Endogâmicos F344 , Ratos Mutantes , Análise de Sequência , Serina/genética , Análise de Sobrevida , Natação , Treonina/genética , Fatores de Tempo , Xenopus
4.
Biochim Biophys Acta ; 1808(1): 454-60, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20974108

RESUMO

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.


Assuntos
Eletrofisiologia/métodos , Canal de Potássio Kv1.1/efeitos dos fármacos , Canal de Potássio Kv1.1/metabolismo , Mitocôndrias/metabolismo , Animais , Biofísica/métodos , Encéfalo/metabolismo , Membrana Celular/metabolismo , Charibdotoxina/química , Peptídeos/química , Fosfatidilcolinas/química , Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo
5.
Bioorg Med Chem ; 16(6): 3067-75, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18226531

RESUMO

Kv1.1 channels are expressed in many regions of the brain and spinal cord [Monaghan, M. M.; Trimmer, J. S.; Rhodes, K. J. J. Neurosci.2001, 21, 5973; Rasband, M. N.; Trimmer, J. S. J. Comp. Neurol.2001, 429, 166; Trimmer, J. S.; Rhodes, K. J. Ann. Rev. Physiol.2004, 66, 477]. When expressed alone, they produce a delayed rectifier slowly inactivating type current that contributes to hyperpolarizing the neuron following depolarization. In the hippocampus Kv1.1 is co-expressed with Kvbeta1 (and other beta subunits), which converts Kv1.1 into a transient, fast inactivating current, reducing its ability to hyperpolarize the cell and thus increasing neuronal excitability. To reduce neuronal excitability, screening for compounds that prevent inactivation of Kv1.1 channels by Kvbeta1 was performed using a yeast two-hybrid screen. A variety of compounds were discovered in this assay and subsequently determined to disrupt inactivation of the ionic currents, and hence were termed 'disinactivators'. Several of these disinactivators also inhibited pentylenetetrazole-induced seizures (PTZ) in mice. Compounds were found to act by several mechanisms to prevent Kvbeta1 inactivation of Kv1.1 channels, including enhancement of Ca(2+) release/influx and by direct mechanisms. Two structural classes were identified that act on a Kvbeta1N70-Kv1.1 chimera where the N-terminal 70 amino acids of Kvbeta1 were attached to the N-terminus of Kv1.1. It is likely that these disinactivators act directly on the Kvbeta1 N-terminus or its receptor site on Kv1.1, thus preventing it from blocking Kv1.1 channels. Compounds acting by this mechanism may be useful for reducing neuronal hyperexcitability in diseases such as epilepsy and neuropathic pain.


Assuntos
Canal de Potássio Kv1.1/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Bibliotecas de Moléculas Pequenas , Animais , Cálcio/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Convulsões/prevenção & controle , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-Híbrido
6.
Toxicol Appl Pharmacol ; 226(3): 225-35, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17961620

RESUMO

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K(+) current (I(A)) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 microM to 100 microM, mefenamic acid reversibly inhibited I(A) in a dose-dependent manner. However, mefenamic acid at a concentration of 1 microM significantly increased the amplitude of I(A) to 113+/-1.5% of the control. At more than 10 microM, mefenamic acid inhibited the amplitude of I(A) without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I(A), but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I(A) channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Córtex Cerebelar/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/toxicidade , Canal de Potássio Kv1.1/efeitos dos fármacos , Ácido Mefenâmico/toxicidade , Neurônios/efeitos dos fármacos , Animais , Ácido Araquidônico/toxicidade , Células Cultivadas , Córtex Cerebelar/metabolismo , Relação Dose-Resposta a Droga , Canal de Potássio Kv1.1/fisiologia , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
7.
J Neurophysiol ; 96(3): 1203-14, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16672305

RESUMO

Low threshold, voltage-gated potassium currents (Ikl) are widely expressed in auditory neurons that can fire temporally precise action potentials (APs). In the medial nucleus of the trapezoid body (MNTB), channels containing the Kv1.1 subunit (encoded by the Kcna1 gene) underlie Ikl. Using pharmacology, genetics and whole cell patch-clamp recordings in mouse brain slices, we tested the role of Ikl in limiting AP latency-variability (jitter) in response to trains of single inputs at moderate to high stimulation rates. With dendrotoxin-K (DTX-K, a selective blocker of Kv1.1-containing channels), we blocked Ikl maximally (approximately 80% with 100 nM DTX-K) or partially (approximately 50% with 1-h incubation in 3 nM DTX-K). Ikl was similar in 3 nM DTX-K-treated cells and cells from Kcna1(-/-) mice, allowing a comparison of these two different methods of Ikl reduction. In response to current injection, Ikl reduction increased the temporal window for AP initiation and increased jitter in response to the smallest currents that were able to drive APs. While 100 nM DTX-K caused the largest increases, latency and jitter in Kcna1(-/-) cells and in 3 nM DTX-K-treated cells were similar to each other but increased compared with +/+. The near-phenocopy of the Kcna1(-/-) cells with 3 nM DTX-K shows that acute blockade of a subset of the Kv1.1-containing channels is functionally similar to the chronic elimination of all Kv1.1 subunits. During rapid stimulation (100-500 Hz), Ikl reduction increased jitter in response to both large and small inputs. These data show that Ikl is critical for maintaining AP temporal precision at physiologically relevant firing rates.


Assuntos
Potenciais de Ação/fisiologia , Encéfalo/fisiologia , Canal de Potássio Kv1.1/fisiologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Técnicas In Vitro , Canal de Potássio Kv1.1/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Canais de Potássio/fisiologia
8.
J Neurophysiol ; 95(4): 2032-41, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16306173

RESUMO

The basolateral amygdala (BLA) is the major amygdaloid nucleus distributed with mu opioid receptors. The afferent input from the BLA to the central nucleus of the amygdala (CeA) is considered important for opioid analgesia. However, little is known about the effect of mu opioids on synaptic transmission in the BLA. In this study, we examined the effect of mu opioid receptor stimulation on the inhibitory and excitatory synaptic inputs to CeA-projecting BLA neurons. BLA neurons were retrogradely labeled with a fluorescent tracer injected into the CeA of rats. Whole cell voltage-clamp recordings were performed on labeled BLA neurons in brain slices. The specific mu opioid receptor agonist, (D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO, 1 microM), significantly reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs) in 77% of cells tested. DAMGO also significantly decreased the peak amplitude of evoked IPSCs in 75% of cells examined. However, DAMGO did not significantly alter the frequency of mEPSCs or the peak amplitude of evoked EPSCs in 90% and 75% of labeled cells, respectively. Bath application of the Kv channel blockers, 4-AP (Kv1.1, 1.2, 1.3, 1.5, 1.6, 3.1, 3.2), alpha-dendrotoxin (Kv1.1, 1.2, 1.6), dendrotoxin-K (Kv1.1), or tityustoxin-Kalpha (Kv1.2) each blocked the inhibitory effect of DAMGO on mIPSCs. Double immunofluorescence labeling showed that some of the immunoreactivities of Kv1.1 and Kv1.2 were colocalized with synaptophysin in the BLA. This study provides new information that activation of presynaptic mu opioid receptors primarily attenuates GABAergic synaptic inputs to CeA-projecting neurons in the BLA through a signaling mechanism involving Kv1.1 and Kv1.2 channels.


Assuntos
Tonsila do Cerebelo/fisiologia , Canal de Potássio Kv1.1/fisiologia , Canal de Potássio Kv1.2/fisiologia , Neurônios Aferentes/fisiologia , Receptores Opioides mu/fisiologia , Ácido gama-Aminobutírico/fisiologia , Tonsila do Cerebelo/química , Analgésicos Opioides/farmacologia , Animais , Bicuculina/farmacologia , Venenos Elapídicos/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina/farmacologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Canal de Potássio Kv1.1/análise , Canal de Potássio Kv1.1/efeitos dos fármacos , Canal de Potássio Kv1.2/análise , Canal de Potássio Kv1.2/efeitos dos fármacos , Sistema Límbico/fisiologia , Masculino , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Opioides/fisiologia , Receptores Opioides mu/agonistas , Venenos de Escorpião/farmacologia , Transdução de Sinais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Sinaptofisina/análise , Receptor de Nociceptina
9.
Brain Res ; 1058(1-2): 30-43, 2005 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-16153617

RESUMO

Kv1.1 channels are brain glycoproteins that play an important role in repolarization of action potentials. In previous work, we showed that lack of N-glycosylation, particularly lack of sialylation, of Kv1.1 affected its macroscopic gating properties and slowed activation and C-type inactivation kinetics and produced a depolarized shift in the steady-state activation curve. In our current study, we used single channel analysis to investigate voltage-independent C-type inactivation in both Kv1.1 and Kv1.1N207Q, a glycosylation mutant. Both channels underwent brief and long-lived closures, and the lifetime and frequency of the long-lived closed states were voltage-independent and similar for both channels. We found that, as in macroscopic measurements, Kv1.1N207Q exhibited a approximately 8 mV positive shift in its single channel fractional open time (fo) and a shallower fo-voltage slope compared with Kv1.1. Data suggested that C-type inactivation reflected the equilibration time with at least two slow voltage-independent long-lived closed states that followed the rapid activation process. In addition, data simulation indicated that the C-type inactivation process reflected the equilibration time between the open state and at least two long-lived closed states. Moreover, the faster macroscopic current decay in Kv1.1 mostly reflected a slower equilibration time in these channels as compared with Kv1.1N207Q. Finally, action potential simulations indicated that the N207Q mutation broaden the action potential and decreased the interspike interval. The shape of the action potential was not significantly affected by C-type inactivation, however, for a given channel, C-type inactivation increased the interspike interval. Data and simulations suggested that excitable cells could use differences in K(+) channel glycosylation degree as an additional mechanism to increase channel functional diversity which could modify cell excitability.


Assuntos
Potenciais de Ação/genética , Membrana Celular/metabolismo , Ativação do Canal Iônico/genética , Canal de Potássio Kv1.1/metabolismo , Neurônios/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Simulação por Computador , Cricetinae , Cricetulus , Glicosilação , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Potássio Kv1.1/efeitos dos fármacos , Canal de Potássio Kv1.1/genética , Mutação/genética , Neurônios/efeitos dos fármacos , Processamento de Sinais Assistido por Computador , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA