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1.
Mol Cell Biol ; 38(19)2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29987189

RESUMO

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein that crosses the blood-brain barrier, binds to myelin, and, hence, has been suggested to be a putative agent for the onset of multiple sclerosis, a demyelinating neuroinflammatory disease. Recently, myelin and lymphocyte (MAL) protein has been identified to be a key protein in the cytotoxic effect of Etx; however, the association of Etx with the immune system remains a central question. Here, we show that Etx selectively recognizes and kills only human cell lines expressing MAL protein through a direct Etx-MAL protein interaction. Experiments on lymphocytic cell lines revealed that MAL protein-expressing T cells, but not B cells, are sensitive to Etx and reveal that the toxin may be used as a molecular tool to distinguish subpopulations of lymphocytes. The overall results open the door to investigation of the role of Etx and Clostridium perfringens on inflammatory and autoimmune diseases like multiple sclerosis.


Assuntos
Toxinas Bacterianas/toxicidade , Clostridium perfringens/patogenicidade , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/toxicidade , Células HeLa , Humanos , Células Jurkat , Linfócitos/patologia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia
2.
J Phys Chem Lett ; 9(14): 3800-3807, 2018 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-29924934

RESUMO

Understanding the molecular mechanisms governing protein-nucleic acid interactions is fundamental to many nuclear processes. However, how nucleic acid binding affects the conformation and dynamics of the substrate protein remains poorly understood. Here we use a combination of single molecule force spectroscopy AFM and biochemical assays to show that the binding of TG-rich ssDNA triggers a mechanical switch in the RRM1 domain of TDP-43, toggling between an entropic spring devoid of mechanical stability and a shock absorber bound-form that resists unfolding forces of ∼40 pN. The fraction of mechanically resistant proteins correlates with an increasing length of the TG n oligonucleotide, demonstrating that protein mechanical stability is a direct reporter of nucleic acid binding. Steered molecular dynamics simulations on related RNA oligonucleotides reveal that the increased mechanical stability fingerprinting the holo-form is likely to stem from a unique scenario whereby the nucleic acid acts as a "mechanical staple" that protects RRM1 from mechanical unfolding. Our approach highlights nucleic acid binding as an effective strategy to control protein nanomechanics.


Assuntos
Proteínas de Ligação a DNA/química , Genes de Troca , Proteínas Supressoras de Tumor/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Fenômenos Mecânicos , Domínios Proteicos
3.
eNeuro ; 4(4)2017.
Artigo em Inglês | MEDLINE | ID: mdl-28798954

RESUMO

ε-Toxin is a pore forming toxin produced by Clostridium perfringens types B and D. It is synthesized as a less active prototoxin form that becomes fully active upon proteolytic activation. The toxin produces highly lethal enterotoxaemia in ruminants, has the ability to cross the blood-brain barrier (BBB) and specifically binds to myelinated fibers. We discovered that the toxin induced a release of ATP from isolated mice optic nerves, which are composed of myelinated fibers that are extended from the central nervous system. We also investigated the effect of the toxin on compound action potentials (CAPs) in isolated mice optic nerves. When nerves were stimulated at 100 Hz during 200 ms, the decrease of the amplitude and the area of the CAPs was attenuated in the presence of ε-toxin. The computational modelling of myelinated fibers of mouse optic nerve revealed that the experimental results can be mimicked by an increase of the conductance of myelin and agrees with the pore forming activity of the toxin which binds to myelin and could drill it by making pores. The intimate ultrastructure of myelin was not modified during the periods of time investigated. In summary, the acute action of the toxin produces a subtle functional impact on the propagation of the nerve action potential in myelinated fibers of the central nervous system with an eventual desynchronization of the information. These results may agree with the hypothesis that the toxin could be an environmental trigger of multiple sclerosis (MS).


Assuntos
Potenciais de Ação/efeitos dos fármacos , Toxinas Bacterianas/farmacologia , Nervo Óptico/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Clostridium perfringens/química , Simulação por Computador , Estimulação Elétrica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Nervo Óptico/ultraestrutura , Compostos de Fósforo/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Fatores de Tempo
4.
Biophys J ; 111(7): 1429-1443, 2016 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-27705766

RESUMO

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.


Assuntos
Ânions/metabolismo , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Ânions/química , Carbenoxolona/química , Carbenoxolona/farmacologia , Espaço Extracelular/química , Espaço Extracelular/efeitos dos fármacos , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Técnicas In Vitro , Cinética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Membrana/genética , Neurotransmissores/química , Neurotransmissores/farmacologia , Oócitos/química , Oócitos/metabolismo , Concentração Osmolar , Permeabilidade , Multimerização Proteica , Relação Estrutura-Atividade , Taurina/química , Taurina/metabolismo , Água/química , Xenopus
5.
J Biol Chem ; 291(33): 17197-208, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27261461

RESUMO

Protein misfolding is implicated in neurodegenerative diseases such as ALS, where mutations of superoxide dismutase 1 (SOD1) account for about 20% of the inherited mutations. Human SOD1 (hSOD1) contains four cysteines, including Cys(57) and Cys(146), which have been linked to protein stability and folding via forming a disulfide bond, and Cys(6) and Cys(111) as free thiols. But the roles of the cellular oxidation-reduction (redox) environment in SOD1 folding and aggregation are not well understood. Here we explore the effects of cellular redox systems on the aggregation of hSOD1 proteins. We found that the known hSOD1 mutations G93A and A4V increased the capability of the thioredoxin and glutaredoxin systems to reduce hSOD1 compared with wild-type hSOD1. Treatment with inhibitors of these redox systems resulted in an increase of hSOD1 aggregates in the cytoplasm of cells transfected with mutants but not in cells transfected with wild-type hSOD1 or those containing a secondary C111G mutation. This aggregation may be coupled to changes in the redox state of the G93A and A4V mutants upon mild oxidative stress. These results strongly suggest that the thioredoxin and glutaredoxin systems are the key regulators for hSOD1 aggregation and may play critical roles in the pathogenesis of ALS.


Assuntos
Esclerose Amiotrófica Lateral , Estresse Oxidativo , Agregação Patológica de Proteínas , Dobramento de Proteína , Superóxido Dismutase-1 , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Animais , Linhagem Celular Tumoral , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Humanos , Mutação de Sentido Incorreto , Oxirredução , Agregação Patológica de Proteínas/enzimologia , Agregação Patológica de Proteínas/genética , Ratos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
6.
Neuropharmacology ; 105: 241-257, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26801076

RESUMO

Previous studies documented a cross-talk between purinergic P2X (P2XR) and nicotinic acetylcholine receptors (nAChR) in heterologous expression systems and peripheral preparations. We now investigated if this occurred in native brain preparations and probed its physiological function. We found that P2XR and nAChR were enriched in hippocampal terminals, where both P2X1-3R and α3, but not α4, nAChR subunits were located in the active zone and in dopamine-ß-hydroxylase-positive hippocampal terminals. Notably, P2XR ligands displaced nAChR binding and nAChR ligands displaced P2XR binding to hippocampal synaptosomes. In addition, a negative P2XR/nAChR cross-talk was observed in the control of the evoked release of noradrenaline from rat hippocampal synaptosomes, characterized by a less-than-additive facilitatory effect upon co-activation of both receptors. This activity-dependent cross-inhibition was confirmed in Xenopus oocytes transfected with P2X1-3Rs and α3ß2 (but not α4ß2) nAChR. Besides, P2X2 co-immunoprecipitated α3ß2 (but not α4ß2) nAChR, both in HEK cells and rat hippocampal membranes indicating that this functional interaction is supported by a physical association between P2XR and nAChR. Moreover, eliminating extracellular ATP with apyrase in hippocampal slices promoted the inhibitory effect of the nAChR antagonist tubocurarine on noradrenaline release induced by high- but not low-frequency stimulation. Overall, these results provide integrated biochemical, pharmacological and functional evidence showing that P2X1-3R and α3ß2 nAChR are physically and functionally interconnected at the presynaptic level to control excessive noradrenergic terminal activation upon intense synaptic firing in the hippocampus.


Assuntos
Hipocampo/crescimento & desenvolvimento , Hipocampo/fisiologia , Canais Iônicos/fisiologia , Receptores Nicotínicos/fisiologia , Receptores Pré-Sinápticos/fisiologia , Receptores Purinérgicos P2X/fisiologia , Animais , Dopamina beta-Hidroxilase/metabolismo , Células HEK293 , Humanos , Masculino , Terminações Nervosas/metabolismo , Norepinefrina/metabolismo , Oócitos , Ratos , Ratos Wistar , Receptor Cross-Talk/fisiologia , Receptores Purinérgicos P2X1/fisiologia , Receptores Purinérgicos P2X2/fisiologia , Receptores Purinérgicos P2X3/fisiologia , Sinaptossomos/metabolismo , Xenopus , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
7.
J Biol Chem ; 289(39): 26722-32, 2014 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-25096579

RESUMO

Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.


Assuntos
Esclerose Amiotrófica Lateral/enzimologia , Cisteína/química , Dissulfetos/química , Mutação de Sentido Incorreto , Desdobramento de Proteína , Superóxido Dismutase/química , Substituição de Aminoácidos , Esclerose Amiotrófica Lateral/genética , Cisteína/genética , Humanos , Oxirredução , Estrutura Secundária de Proteína , Superóxido Dismutase/genética , Superóxido Dismutase-1
8.
Am J Physiol Endocrinol Metab ; 306(12): E1354-66, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24735890

RESUMO

The existence of functional connexin36 (Cx36) hemichannels in ß-cells was investigated in pancreatic islets of rat and wild-type (Cx36(+/+)), monoallelic (Cx36(+/-)), and biallelic (Cx36(-/-)) knockout mice. Hemichannel opening by KCl depolarization was studied by measuring ATP release and changes of intracellular ATP (ADP). Cx36(+/+) islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36(-/-) than Cx36(+/+) islets and was not decreased by KCl depolarization; Cx36(+/-) islets showed values between that of control and homozygous islets. Five minimolar extracellular ATP increased ATP content and ATP/ADP ratio and induced a biphasic insulin secretion in depolarized Cx36(+/+) and Cx36(+/-) but not Cx36(-/-) islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential, and their activation was inhibited by mefloquine and glucose (IC50 ∼8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet ß-cells might avoid depolarization-induced ATP loss, allowing an optimum increase of the ATP/ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36(+/-) and Cx36(-/-) islets confirms that Cx36 gap junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.


Assuntos
Glicemia/metabolismo , Conexinas/antagonistas & inibidores , Intolerância à Glucose/metabolismo , Hiperglicemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Regulação para Cima , Trifosfato de Adenosina/metabolismo , Animais , Glicemia/análise , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Intolerância à Glucose/sangue , Heterozigoto , Hiperglicemia/etiologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnicas de Cultura de Tecidos , Regulação para Cima/efeitos dos fármacos
9.
Glia ; 61(12): 1976-89, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24123415

RESUMO

Extracellular purines elicit strong signals in the nervous system. Adenosine-5'-triphosphate (ATP) does not spontaneously cross the plasma membrane, and nervous cells secrete ATP by exocytosis or through plasma membrane proteins such as connexin hemichannels. Using a combination of imaging, luminescence and electrophysiological techniques, we explored the possibility that Connexin 32 (Cx32), expressed in Schwann cells (SCs) myelinating the peripheral nervous system could be an important source of ATP in peripheral nerves. We triggered the release of ATP in vivo from mice sciatic nerves by electrical stimulation and from cultured SCs by high extracellular potassium concentration-evoked depolarization. No ATP was detected in the extracellular media after treatment of the sciatic nerve with Octanol or Carbenoxolone, and ATP release was significantly inhibited after silencing Cx32 from SCs cultures. We investigated the permeability of Cx32 to ATP by expressing Cx32 hemichannels in Xenopus laevis oocytes. We found that ATP release is coupled to the inward tail current generated after the activation of Cx32 hemichannels by depolarization pulses, and it is sensitive to low extracellular calcium concentrations. Moreover, we found altered ATP release in mutated Cx32 hemichannels related to the X-linked form of Charcot-Marie-Tooth disease, suggesting that purinergic-mediated signaling in peripheral nerves could underlie the physiopathology of this neuropathy.


Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Carbenoxolona/farmacologia , Conexinas/genética , Estimulação Elétrica , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/genética , Masculino , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Células de Schwann/efeitos dos fármacos , Nervo Isquiático/efeitos dos fármacos , Xenopus laevis
10.
Biochim Biophys Acta ; 1828(1): 69-78, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22326946

RESUMO

Gap junction communication is crucial for myelination and axonal survival in both the peripheral nervous system (PNS) and central nervous system (CNS). This review examines the different types of gap junctions in myelinating glia of the PNS and CNS (Schwann cells and oligodendrocytes respectively), including their functions and involvement in neurological disorders. Gap junctions mediate intercellular communication among Schwann cells in the PNS, and among oligodendrocytes and between oligodendrocytes and astrocytes in the CNS. Reflexive gap junctions mediating transfer between different regions of the same cell promote communication between cellular compartments of myelinating glia that are separated by layers of compact myelin. Gap junctions in myelinating glia regulate physiological processes such as cell growth, proliferation, calcium signaling, and participate in extracellular signaling via release of neurotransmitters from hemijunctions. In the CNS, gap junctions form a glial network between oligodendrocytes and astrocytes. This transcellular communication is hypothesized to maintain homeostasis by facilitating restoration of membrane potential after axonal activity via electrical coupling and the re-distribution of potassium ions released from axons. The generation of transgenic mice for different subsets of connexins has revealed the contribution of different connexins in gap junction formation and illuminated new subcellular mechanisms underlying demyelination and cognitive defects. Alterations in metabolic coupling have been reported in animal models of X-linked Charcot-Marie-Tooth disease (CMTX) and Pelizaeus-Merzbarcher-like disease (PMLD), which are caused by mutations in the genes encoding for connexin 32 and connexin 47 respectively. Future research identifying the expression and regulation of gap junctions in myelinating glia is likely to provide a better understanding of myelinating glia in nervous system function, plasticity, and disease. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.


Assuntos
Junções Comunicantes/metabolismo , Bainha de Mielina/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Comunicação Celular , Permeabilidade da Membrana Celular , Conexinas/genética , Conexinas/metabolismo , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Humanos , Mutação , Bainha de Mielina/patologia , Bainha de Mielina/fisiologia , Neuroglia/metabolismo
11.
Mol Cell Neurosci ; 49(3): 364-74, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22306863

RESUMO

Secreted Protein Acidic and Rich in Cysteine (SPARC) is a matricellular protein produced by glial cells. Although it is highly expressed in synaptogenic areas in the developing nervous system, it is still unclear whether this molecule displays an action on synaptic activity. We show that nanomolar concentrations of SPARC favour a more efficient synapse formation and increase short term depression in single cell cholinergic microcultures. The change in synaptic plasticity, which is also observed when SPARC is locally secreted on stable synapses for 24-48 h, is caused by a high release probability and a reduction in the size of the rapidly releasable pool of vesicles. Both features are attributable to synapses operating at an immature stage as demonstrated by correlative electrophysiology and electron microscopy experiments. Presynaptic terminals developed in the presence of SPARC display few cytoplasmic vesicles and two to threefold decrease in the number of docked vesicles at active zones. At the postsynaptic level, the analysis of miniature excitatory postsynaptic currents suggests SPARC has little effect on the number of nicotinic receptors but might alter their composition. The widespread distribution of SPARC makes current findings potentially relevant to other excitatory synapses and development of neuronal circuits.


Assuntos
Fibras Colinérgicas/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Osteonectina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Animais , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Microscopia Eletrônica , Neuroglia/metabolismo , Plasticidade Neuronal/fisiologia , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura
12.
Neurochem Int ; 59(6): 954-64, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21782871

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the selective loss of both upper and lower motoneurons (MNs). The familial form of the illness is associated with mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD-1) enzyme, but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form of ALS. Although many proposals have been suggested over the years, the mechanisms underlying the characteristic selective killing of MN in ALS remain unknown. In this study we tested the effect of sera from sporadic ALS patients on NMDA receptors (NMDAR). We hypothesize that an endogenous seric factor is implicated in neuronal death in ALS, mediated by the modulation of NMDAR. Sera from ALS patients and from healthy subjects were pretreated to inactivate complement pathways and dialyzed to remove glutamate and glycine. IgGs from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and ionic currents were recorded using the two-electrode voltage clamp technique. Sera from sporadic ALS patients induced transient oscillatory currents in oocytes expressing NMDAR with a significantly higher total electrical charge than that induced by sera from healthy subjects. Sera from patients with other neuromuscular diseases did not exert this effect. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. The PLC inhibitor, U-73122, and the IP(3) receptor antagonist, 2-APB, also inhibited the sera-induced currents. The oscillatory signal recorded was due to internal calcium mobilization. Isolated IgGs from ALS patients significantly affected the activity of oocytes injected with NMDAR, causing a 2-fold increase over the response recorded for IgGs from healthy subjects. Our data support the notion that ALS sera contain soluble factors that mobilize intracellular calcium, not opening directly the ionic conductance, but through the non-canonical activation of NMDAR.


Assuntos
Esclerose Amiotrófica Lateral/sangue , Esclerose Amiotrófica Lateral/fisiopatologia , Proteínas Sanguíneas/toxicidade , Sinalização do Cálcio/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Adulto , Idoso , Animais , Sinalização do Cálcio/fisiologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Ácido Glutâmico/fisiologia , Ácido Glutâmico/toxicidade , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Degeneração Neural/induzido quimicamente , Degeneração Neural/fisiopatologia , Neurotoxinas/toxicidade , Oócitos , Receptores de N-Metil-D-Aspartato/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Xenopus laevis
13.
Cell Calcium ; 49(3): 184-90, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21349580

RESUMO

Amyloid beta (Aß) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aß oligomers cause neurotoxicity remain unknown. We recently reported that Aß oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aß oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aß oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aß oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aß oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aß oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aß damage to neurons in Alzheimers disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Maleato de Dizocilpina/farmacologia , Fenômenos Eletrofisiológicos , Feminino , Humanos , Memantina/farmacologia , Neurônios/metabolismo , Oócitos/metabolismo , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/genética , Valina/análogos & derivados , Valina/farmacologia , Xenopus laevis/embriologia
14.
J Neuroimmunol ; 229(1-2): 157-68, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20800908

RESUMO

Massive programmed cell death (PCD) of developing chick embryo motoneurons (MNs) occurs in a well defined temporal and spatial sequence between embryonic day (E) 6 and E10. We have found that, when administered in ovo, either circulating immunoglobulins G (IgGs) or cerebrospinal fluid from patients with MN disease can rescue a significant number of chick embryo MNs from normally occurring PCD. An increase of branching of intramuscular nerves was also observed that may account for the rescuing effects of pathologic IgGs. Proteomic analysis and further analysis by ELISA indicated that these effects may be mediated by the interaction of circulating human immunoglobulins with proteins of the semaphorin family.


Assuntos
Apoptose/efeitos dos fármacos , Imunoglobulinas/farmacologia , Doença dos Neurônios Motores/imunologia , Neurônios Motores/efeitos dos fármacos , Músculo Esquelético/inervação , Junção Neuromuscular/efeitos dos fármacos , Análise de Variância , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Gânglios Espinais/citologia , Cones de Crescimento/efeitos dos fármacos , Humanos , Imunoglobulinas/imunologia , Técnicas In Vitro , Masculino , Doença dos Neurônios Motores/sangue , Neurônios Motores/citologia , Músculo Esquelético/embriologia , Junção Neuromuscular/fisiologia , Proteômica/métodos , Semaforinas/metabolismo , Soro/química , Soro/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estatística como Assunto , Estatísticas não Paramétricas , Transfecção/métodos , Tubulina (Proteína)/metabolismo
15.
J Biol Chem ; 283(13): 8756-64, 2008 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-18218624

RESUMO

Kv1.3 activity is determined by raft association. In addition to Kv1.3, leukocytes also express Kv1.5, and both channels control physiological responses. Because the oligomeric composition may modify the channel targeting to the membrane, we investigated heterotetrameric Kv1.3/Kv1.5 channel traffic and targeting in HEK cells. Kv1.3 and Kv1.5 generate multiple heterotetramers with differential surface expression according to the subunit composition. FRET analysis and pharmacology confirm the presence of functional hybrid channels. Raft association was evaluated by cholesterol depletion, caveolae colocalization, and lateral diffusion at the cell surface. Immunoprecipitation showed that both Kv1.3 and heteromeric channels associate with caveolar raft domains. However, homomeric Kv1.3 channels showed higher association with caveolin traffic. Moreover, FRAP analysis revealed higher mobility for hybrid Kv1.3/Kv1.5 than Kv1.3 homotetramers, suggesting that heteromers target to distinct surface microdomains. Studies with lipopolysaccharide-activated macrophages further supported that different physiological mechanisms govern Kv1.3 and Kv1.5 targeting to rafts. Our results implicate the traffic and localization of Kv1.3/Kv1.5 heteromers in the complex regulation of immune system cells.


Assuntos
Membrana Celular/metabolismo , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/metabolismo , Animais , Células Cultivadas , Eletrofisiologia , Humanos , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.5/genética , Macrófagos/metabolismo , Camundongos , Técnicas de Patch-Clamp , Ligação Proteica , Transporte Proteico , Ratos
16.
Neurochem Int ; 50(1): 256-63, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17030469

RESUMO

During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme. In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.


Assuntos
Órgão Elétrico/enzimologia , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar , Células HeLa , Humanos , Dados de Sequência Molecular , Pirofosfatases/genética , Torpedo
17.
Biochem Biophys Res Commun ; 352(4): 913-8, 2007 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-17157812

RESUMO

Voltage-dependent K(+) (Kv) channels are involved in the immune response. Kv1.3 is highly expressed in activated macrophages and T-effector memory cells of autoimmune disease patients. Macrophages are actively involved in T-cell activation by cytokine production and antigen presentation. However, unlike T-cells, macrophages express Kv1.5, which is resistant to Kv1.3-drugs. We demonstrate that mononuclear phagocytes express different Kv1.3/Kv1.5 ratios, leading to biophysically and pharmacologically distinct channels. Therefore, Kv1.3-based treatments to alter physiological responses, such as proliferation and activation, are impaired by Kv1.5 expression. The presence of Kv1.5 in the macrophagic lineage should be taken into account when designing Kv1.3-based therapies.


Assuntos
Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Células Cultivadas , Camundongos , Venenos de Escorpião/farmacologia
18.
J Biol Chem ; 281(49): 37675-85, 2006 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-17038323

RESUMO

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.


Assuntos
Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.5/metabolismo , Macrófagos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Feminino , Humanos , Técnicas In Vitro , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Macrófagos/ultraestrutura , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Oócitos/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Xenopus laevis
19.
J Cell Physiol ; 206(1): 95-102, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15965959

RESUMO

ATP is an electrically charged molecule that functions both in the supply of energy necessary for cellular activity and as an intercellular signaling molecule. Although controlled ATP secretion occurs via exocytosis of granules and vesicles, in some cells, and under certain conditions, other mechanisms control ATP release. Gap junctions, intercellular channels formed by connexins that link the cytoplasm of two adjacent cells, control the passage of ions and molecules up to 1 kDa. The channel is formed by two moieties called hemichannels, or connexons, and it has been suggested that these may represent an alternative pathway for ATP release. We have investigated the release of ATP through hemichannels from Xenopus oocytes that are formed by Connexin 38 (Cx38), an endogenous, specific type of connexin. These hemichannels generate an inward current that is reversibly activated by calcium-free solution and inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it is decreased in oocytes injected with an antisense oligonucleotide against Cx38 mRNA (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of these endogenous connexons also allows transfer of Lucifer Yellow. We have found that the release of ATP is coincident with the opening of hemichannels: it is calcium-sensitive, is inhibited by octanol and flufenamic acid, is inhibited in ASCx38 injected oocytes, and is increased by overexpression of Cx38. Taken together, our results suggest that ATP is released through activated hemichannels in Xenopus oocytes.


Assuntos
Trifosfato de Adenosina/metabolismo , Junções Comunicantes/metabolismo , Oócitos/metabolismo , Xenopus laevis , Animais , Anti-Inflamatórios/farmacologia , Cálcio/metabolismo , Conexinas/genética , Conexinas/metabolismo , Feminino , Ácido Flufenâmico/farmacologia , Corantes Fluorescentes/metabolismo , Junções Comunicantes/ultraestrutura , Ionóforos/farmacologia , Isoquinolinas/metabolismo , Nistatina/farmacologia , Octanóis/farmacologia , Oligonucleotídeos Antissenso/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp
20.
An. R. Acad. Farm ; 71(4): 821-833, oct. 2005. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-044379

RESUMO

Corrientes colinérgicas de cerebro humano fueron registradas en oocitos de Xenopus laevis trasplantados con membranas de cerebro humano procedentes de dos zonas diferentes, la corteza frontal y el hipocampo. Las corrientes registradas fueron activadas por el receptor nicotínico o por el receptor nicotínico o muscarínico de la acetilcolina. Se probaron los efectos de diferentes agonistas nicotínicos como acetilcolina, nicotina y yoduro de 1,1-dimetil-4-fenil-piperazinio (DMPP), y antagonistas del receptor nicotínico como a-bungarotoxina y d-tubocurarina en los oocitos transplantados. Detectamos cuatro clases de cinéticas de corrientes nicotínicas. Las diferencias en la amplitud y en la carga eléctrica total de las corrientes provocadas por varios agonistas en el rango de potencial mantenido no fueron significativas, excepto en el caso del DMPP a un potencial mantenido de -90 mV. Nuestros resultados indican que las formas alfa4beta2, alfa3beta4 y alfa7 son los principales receptores nicotínicos en el cerebro humano


Cholinergic human brain currents were recorded in Xenopus laevis oocytes transplanted with human cerebral membranes from two different zones, the frontal cortex and the hippocampus. The recorded currents were supported by the nicotinic or the muscarinic acetylcholine receptor. We tested the effects of a number of several nicotinic agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and the nicotinic receptor antagonists a-bungarotoxin and d-tubocurarine on the transplanted oocytes. We detected four kinds of nicotinic current kinetics. The differences in the amplitude and in the total electric charge of the currents elicited by various agonists at a range of holding potentials were not significant, except in the case of DMPP at a holding potential of -90 mV. Our results indicate that alpha4beta2, alpha3beta4 and alpha7 are the main nicotinic receptors in human brain


Assuntos
Oócitos , Membranas , Xenopus laevis/cirurgia , Acetilcolina/farmacologia , Colinérgicos/química , Colinérgicos/farmacologia , Nicotina/química , Nicotina/farmacologia , Telencéfalo , Química Encefálica , Protocolos , Oócitos/química , Acetilcolina/química , Telencéfalo/ultraestrutura
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