RESUMO
BACKGROUND: Cutaneous malignant melanoma arises from transformed melanocytes de novo or from congenital or acquired melanocytic naevi. We have recently reported that T-type Ca2+ channels (TT-Cs) are upregulated in human melanoma and play an important role in cell proliferation. OBJECTIVES: To describe for the first time in formalin-fixed paraffin-embedded tissue the immunoexpression of TT-Cs in biopsies of normal skin, acquired melanocytic naevi and melanoma, in order to evaluate their role in melanomagenesis and/or tumour progression, their utility as prognostic markers and their possible use in targeted therapies. METHODS: Tissue samples from normal skin, melanocytic naevi and melanoma were subjected to immunohistochemistry for two TT-Cs (Cav3.1, Cav3.2); markers of proliferation (Ki67), the cell cycle (cyclin D1), hypoxia (Glut1), vascularization (CD31) and autophagy (LC3); BRAF V600E mutation (VE1) and phosphatase and tensin homologue (PTEN). Immunostaining was evaluated by histoscore. In silico analysis was used to assess the prognostic value of TT-C overexpression. RESULTS: TT-C immunoexpression increased gradually from normal skin to common naevi, dysplastic naevi and melanoma samples, but with differences in the distribution of both isoforms. Particularly, Cav3.2 expression was significantly higher in metastatic melanoma than in primary melanoma. Statistical correlation showed a linear interaction between PTEN loss/BRAF V600E/Cav3.1/LC3/ Ki67/cyclin D1/Cav3.2/Glut1. Disease-free survival (DFS) and overall survival correlated inversely with overexpression of Cav3.2. DFS also correlated inversely with overexpression of Cav3.1. CONCLUSIONS: TT-C immunoexpression on melanocytic neoplasms is consistent with our previous in vitro studies and appears to be related to tumour progression. TT-C upregulation can be considered as a prognostic marker using The Cancer Genome Atlas database. The high expression of Cav3.2 in metastatic melanoma encourages the investigation of the use of TT-C blockers in targeted therapies.
Assuntos
Biomarcadores Tumorais/metabolismo , Canais de Cálcio Tipo T/metabolismo , Melanoma/diagnóstico , Nevo Pigmentado/diagnóstico , Neoplasias Cutâneas/diagnóstico , Proliferação de Células/fisiologia , Progressão da Doença , Intervalo Livre de Doença , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Melanoma/mortalidade , Recidiva Local de Neoplasia/etiologia , Nevo Pigmentado/mortalidade , Prognóstico , Neoplasias Cutâneas/mortalidade , Regulação para CimaRESUMO
[This corrects the article DOI: 10.1155/2015/587135.].
RESUMO
The remodeling of Ca(2+) signaling is a common finding in cancer pathophysiology serving the purpose of facilitating proliferation, migration, or survival of cancer cells subjected to stressful conditions. One particular facet of these adaptive changes is the alteration of Ca(2+) fluxes through the plasma membrane, as described in several studies. In this review, we summarize the current knowledge about the expression of different Ca(2+) channels in the plasma membrane of melanoma cells and its impact on oncogenic Ca(2+) signaling. In the last few years, new molecular components of Ca(2+) influx pathways have been identified in melanoma cells. In addition, new links between Ca(2+) homeostasis and specific cell processes important in melanoma tumor progression have been unveiled. Thus, not only do Ca(2+) channels appear to have a potential as prognostic markers, but their pharmacological blockade or gene silencing is hinted as interesting therapeutic approaches.
Assuntos
Bloqueadores dos Canais de Cálcio/administração & dosagem , Canais de Cálcio/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Animais , Antineoplásicos/administração & dosagem , Sinalização do Cálcio/efeitos dos fármacos , Humanos , Modelos Biológicos , Terapia de Alvo Molecular/métodosRESUMO
The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.
Assuntos
Canais de Cálcio/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/metabolismo , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citometria de Fluxo , Fura-2/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Manganês/metabolismo , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Mibefradil/farmacologia , Imagem Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
All auxiliary alpha2delta subunits of voltage-gated Ca2+ (Ca(V)) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in alpha2delta-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the alpha2delta-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant alpha2delta-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT alpha2delta-2, the MIDAS mutant alpha2delta-2 subunit did not enhance and, in some cases, further diminished Ca(V)1.2, -2.1, and -2.2 currents coexpressed with beta1b by using either Ba2+ or Na+ as a permeant ion. Furthermore, expression of the MIDAS mutant alpha2delta-2 reduced surface expression and strongly increased the perinuclear retention of Ca(V)alpha1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108-15 cells. Despite the presence of endogenous alpha2delta subunits, heterologous expression of alpha2delta-2 in differentiated NG108-15 cells further enhanced the endogenous high-threshold Ca2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that alpha2delta subunits normally interact with the Ca(V)alpha1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the alpha2delta subunit is required to promote trafficking of the alpha1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.
Assuntos
Canais de Cálcio/metabolismo , Modelos Moleculares , Fator de von Willebrand/metabolismo , Sítios de Ligação , Canais de Cálcio/química , Canais de Cálcio/genética , Cátions Bivalentes/metabolismo , DNA Complementar/genética , Eletrofisiologia , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Metais/metabolismo , Microscopia Confocal , Mutação/genética , Conformação ProteicaRESUMO
Voltage-dependent Ca(2+) channels (VDCCs) are subject to modulation by a number of pathways, including membrane-delimited inhibition by heterotrimeric G-proteins and modulation through phosphorylation by diverse kinases. Here we report that in the Xenopus oocyte expression system Ca(V)2.2 channels undergo a sustained, linear and irreversible run-up lasting up to 30 min, which is potentiated during G-protein-mediated inhibition by activation of co-expressed G-protein coupled receptors (GPCRs). This up-regulation is not a result of receptor desensitization, but is associated with a hyperpolarization of the voltage for activation and depends on the presence of accessory subunits such that beta subunits promote, and alpha2delta subunits oppose the current increase. We have investigated the involvement of G-proteins and found that over-expression of Galpha(o) subunits or Galpha-transducin reduced the amount of agonist-mediated up-regulation. However, we have found no evidence for the involvement of any second messenger pathways in the increase of current run-up in the presence of a GPCR agonist. Taken together, our data suggest that the effect reported herein involves an enhancement of the GTPase activity of endogenous Galpha subunits, which is triggered by GPCR activation and mediated by accessory Ca(V)beta subunits. It may involve an increased association of Ca(V)beta subunits with alpha1 subunits in the plasma membrane or trafficking of channels to the plasma membrane.
Assuntos
Canais de Cálcio/biossíntese , Receptores de Dopamina D2/metabolismo , Regulação para Cima/fisiologia , Animais , Agonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Receptores de Dopamina D2/agonistas , Regulação para Cima/efeitos dos fármacos , Xenopus laevisRESUMO
We present the case of a 76 year-old man, intervened of an obstruction bilateral iliac by means of placement of a prosthesis aortobifemoral that presented pain in the grave left iliac and fever in needles of 39 degrees C to the five years of the intervention. In the physical exploration it highlighted a painful abdomen in the grave left iliac with signs of peritoneal irritation. In the laboratory tests a leukocytosis was detected with neutrophilia and negative culture. The computed thomography (CT) show the presence of gas bubbles around the prosthesis, as well as a liquid collection with areas necrotics in their interior that affected to the psoas and iliac muscles. In the same exploration the aspirative puncture with drainage of the absces demonstrated in the cultivations carried out in aerobic means the presence of Enterococcus faecalis and Enterobacter cloacae. When presenting a high gastrointestinal hemorrhage abruptly, he was practiced and gastroduodenal endoscope in which a aortoduodenal fistula was evidenced with having bled active. When a bypass extra-anatomic, the sick person will practice it died when presenting a shock abrupt hipovolemic that he didn't respond to the pertinent treatment. We analyze the approaches current diagnoses of infection of the vascular prosthesis and their more serious complication, the aortoenteric fistula (AEF) that either appears in the 0.3-5.9% of the patients who undergo prosthetic reconstruction of the abdominal aorta, for occlusive or aneurismal disease. We highlight the importance of carrying out a precocious diagnosis of the infection of the portion retroperitoneal of the vascular graft that, often, it is manifested with subtle and not specific clinical signs, with the techniques at the moment available as: the CT, fine needle aspiration guided by her, and to diminish the rates of mortality, from the current of 43%, until the most optimistic estimated in 19%.
Assuntos
Aorta Abdominal/cirurgia , Doenças da Aorta/etiologia , Prótese Vascular/efeitos adversos , Duodenopatias/etiologia , Enterococcus , Veia Femoral/cirurgia , Infecções por Bactérias Gram-Positivas/complicações , Fístula Intestinal/etiologia , Infecções Relacionadas à Prótese/complicações , Fístula Vascular/etiologia , Idoso , Humanos , MasculinoRESUMO
beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Ativação do Canal Iônico , Animais , Canais de Cálcio/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Agonistas de Dopamina/farmacologia , Condutividade Elétrica , Expressão Gênica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Mutação , Oligonucleotídeos Antissenso/genética , Oócitos/metabolismo , Ligação Proteica , Subunidades Proteicas , Quimpirol/farmacologia , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Xenopus laevisRESUMO
The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.
Assuntos
Ataxia/genética , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Epilepsia/genética , Células de Purkinje/metabolismo , Animais , Ataxia/complicações , Encéfalo/metabolismo , Encéfalo/patologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Mapeamento Cromossômico , Eletroencefalografia , Epilepsia/complicações , Homozigoto , Hibridização In Situ , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Subunidades Proteicas , Células de Purkinje/patologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , XenopusRESUMO
Bis(7)-tacrine is a potent acetylcholinesterase inhibitor in which two tacrine molecules are linked by a heptylene chain. We tested the effects of bis(7)-tacrine on the spontaneous synaptic activity. Miniature endplate potentials (MEPPs) were recorded extracellularly on slices of electric organ of Torpedo marmorata. Bis(7)-tacrine, at a concentration of 100 nM, increased the magnitudes that describe MEPPs: amplitude, area, rise time, rate of rise, and half-width. We also tested the effect of bis(7)-tacrine on nicotinic acetylcholine receptors by analyzing the currents elicited by acetylcholine (100 microM) in Torpedo electric organ membranes transplanted in Xenopus laevis oocytes. Bis(7)-tacrine inhibited the acetylcholine-induced currents in a reversible manner (IC(50) = 162 nM). The inhibition of nicotinic acetylcholine receptors was not voltage dependent, and bis(7)-tacrine increased the desensitization of nicotinic acetylcholine receptors. The Hill coefficient for bis(7)-tacrine was -0.72 +/- 0.02, indicating that bis(7)-tacrine binds to the nicotinic acetylcholine receptor in a molecular ratio of 1:1, but does not affect the binding of alpha-bungarotoxin with the nicotinic acetylcholine receptor. In conclusion, bis(7)-tacrine greatly increases the spontaneous quantal release from peripheral cholinergic terminals at a much lower concentration than tacrine. Bis(7)-tacrine also blocks acetylcholine-induced currents of Torpedo electric organ, although the mechanism is different from that of tacrine: bis(7)-tacrine enhances desensitization, whereas tacrine reduces it.
Assuntos
Inibidores da Colinesterase/farmacologia , Órgão Elétrico/fisiologia , Receptores Nicotínicos/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Tacrina/análogos & derivados , Tacrina/farmacologia , Animais , Bungarotoxinas/metabolismo , Bungarotoxinas/farmacologia , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Órgão Elétrico/efeitos dos fármacos , Feminino , Radioisótopos do Iodo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/metabolismo , Oócitos/fisiologia , Oócitos/transplante , Receptores Nicotínicos/metabolismo , Solubilidade , Tacrina/química , Torpedo , Xenopus laevisRESUMO
The accessory beta subunits of voltage-dependent Ca2+ channels (VDCCs) have been shown to regulate their biophysical properties and have also been suggested to antagonise the G protein inhibition of N-type (alpha1B), P/Q-type (alpha1A) and alpha1E channels. Here we have examined the voltage-dependent involvement of the four neuronal isoforms (beta1b, beta2a, beta3 and beta4) in the process of G protein modulation of alpha1B Ca2+ channels. All beta subunits hyperpolarized alpha1B current activation, and all antagonised the G protein-mediated depolarisation of current activation. However, except in the case of beta2a, there was no generalised reduction by beta subunits in the maximal extent of receptor-mediated inhibition of alpha1B current. In addition, all VDCC beta subunits enhanced the rate of current facilitation at +100 mV, for both receptor-mediated and tonic modulation. The rank order for enhancement of facilitation rate was beta3 > beta4 > beta1b > beta2a. In contrast, the amount of voltage-dependent facilitation during tonic modulation was reduced by beta subunit co-expression, despite the fact that the apparent Gbetagamma dissociation rate at +100 mV was enhanced by beta subunits to a similar level as for agonist-induced modulation. Our data provide evidence that G protein activation antagonises Ca2+-channel beta subunit-induced hyperpolarisation of current activation. Conversely, co-expression of all beta subunits increases the apparent Gbetagamma dimer dissociation rate during a depolarising prepulse. This latter feature suggests the co-existence of bound Ca2+-channel beta subunits and Gbetagamma dimers on the alpha1B subunits. Future work will determine how the interaction between Gbetagamma dimers and Ca2+-channel beta subunits with alpha1B results in a functional antagonism at the molecular level.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Animais , Canais de Cálcio/genética , DNA/genética , Agonistas de Dopamina/farmacologia , Eletrofisiologia , Proteínas de Ligação ao GTP/agonistas , Proteínas de Ligação ao GTP/antagonistas & inibidores , Cinética , Potenciais da Membrana/fisiologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Quimpirol/farmacologia , Ratos , Receptores de Dopamina D2/efeitos dos fármacos , Xenopus laevisRESUMO
Voltage-dependent calcium channels consist of a pore-forming transmembrane alpha1-subunit, which is known to associate with a number of accessory subunits, including alpha2-delta- and beta-subunits. The beta-subunits, of which four have been identified (beta1-4), are intracellular proteins that have marked effects on calcium channel trafficking and function. In a previous study, we observed that the beta1b-subunit showed selective plasma membrane association when expressed alone in COS7 cells, whereas beta3 and beta4 did not. In this study, we have examined the basis for this, and have identified, by making chimeric beta-subunits, that the C-terminal region, which shows most diversity between beta-subunits, of beta1b is responsible for its plasma membrane association. Furthermore we have identified, by deletion mutations, an 11-amino acid motif present in the C terminus of beta1b but not in beta3 (amino acids 547-556 of beta1b, WEEEEDYEEE), which when deleted, reduces membrane association of beta1b. Future research aims to identify what is binding to this sequence in beta1b to promote membrane association of this calcium channel subunit. It is possible that such membrane association is important for the selective localization or clustering of particular calcium channels with which beta1b is associated.
Assuntos
Canais de Cálcio/genética , Animais , Células COS , Canais de Cálcio/biossíntese , Membrana Celular/fisiologia , Núcleo Celular/fisiologia , Núcleo Celular/ultraestrutura , Quimera/genética , Chlorocebus aethiops , DNA/biossíntese , DNA/genética , Cães , Eletrofisiologia , Deleção de Genes , Imuno-Histoquímica , Rim/citologia , Mutação/genética , Mutação/fisiologia , Oócitos/metabolismo , Transfecção/genética , XenopusRESUMO
To examine the role of the intracellular N terminus in the G-protein modulation of the neuronal voltage-dependent calcium channel (VDCC) alpha1B, we have pursued two routes of investigation. First, we made chimeric channels between alpha1B and alpha1C, the latter not being modulated by Gbeta gamma subunits. VDCC alpha1 subunit constructs were coexpressed with accessory alpha2delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. G-protein modulation of expressed alpha1 subunits was induced by activation of coexpressed dopamine (D2) receptors with quinpirole in oocytes, or by cotransfection of Gbeta1gamma2 subunits in COS-7 cells. For the chimeric channels, only those with the N terminus of alpha1B showed any G-protein modulation; further addition of the first transmembrane domain and I-II intracellular linker of alpha1B increased the degree of modulation. To determine the amino acids within the alpha1B N terminus, essential for G-protein modulation, we made mutations of this sequence and identified three amino acids (S48, R52, and R54) within an 11 amino acid sequence as being critical for G-protein modulation, with I49 being involved to a lesser extent. This sequence may comprise an essential part of a complex Gbeta gamma-binding site or be involved in its subsequent action.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Fragmentos de Peptídeos/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Deleção Cromossômica , Agonistas de Dopamina/farmacologia , Feminino , Proteínas de Ligação ao GTP/química , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Técnicas de Patch-Clamp , Mutação Puntual , Receptores de Dopamina D2/agonistas , Proteínas Recombinantes de Fusão/metabolismo , XenopusRESUMO
The molecular determinants for G-protein regulation of neuronal calcium channels remain controversial. We have generated a series of alpha 1B/alpha 1E chimeric channels, since rat brain alpha 1E (rbEII), unlike human alpha 1E, showed no G-protein modulation. The study, carried out in parallel using D2 receptor modulation of calcium currents in Xenopus oocytes of G beta gamma modulation of calcium currents in COS-7 cells, consistently showed an essential role for domain I (from the N terminus to the end of the I-II loop) of the alpha 1B Ca2+ channel in G-protein regulation, with no additional effect of the C terminal of alpha 1B. The I-II loop alone of alpha 1B, or the I-II loop together with the C-terminal tail, was insufficient to confer G-protein modulation of alpha 1E (rbEII). We have further observed that the alpha 1E clone rbEII is truncated at the N-terminus compared to other alpha 1 subunits, and we isolated a PCR product from rat brain equivalent to a longer N-terminal isoform. The long N-terminal alpha 1E, unlike the short form, showed G-protein modulation. Furthermore, the equivalent truncation of alpha 1B (delta N1-55) abolished G-protein modulation of alpha 1B. Thus, we propose that the N terminus of alpha 1B and alpha 1E calcium channels contains essential molecular determinants for membrane-delimited G-protein inhibition, and that other regions, including the I-II loop and the C terminus, do not play a conclusive role alone.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Baclofeno/farmacologia , Sítios de Ligação , Canais de Cálcio/genética , Células Cultivadas , Eletrofisiologia , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Oócitos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , XenopusRESUMO
1. We studied the G protein inhibition of heteromultimeric neuronal Ca2+ channels by constructing a series of chimeric channels between the strongly modulated alpha1B subunit and the alpha1E(rbEII) subunit, which showed no modulation. 2. In parallel studies, alpha1 subunit constructs were co-expressed together with the accessory Ca2+ channel alpha2-delta and beta2a subunits in mammalian (COS-7) cells and Xenopus oocytes. G protein inhibition of expressed Ca2+ channel currents was induced by co-transfection of Gbeta1 and Ggamma2 subunits in COS-7 cells or activation of co-expressed dopamine (D2) receptors by quinpirole (100 nM) in oocytes. 3. The data indicate that transfer of the alpha1B region containing the N-terminal, domain I and the I-II loop (i.e. the alpha1B1-483 sequence), conferred G protein modulation on alpha1E(rbEII), both in terms of a slowing of activation kinetics and a reduction in current amplitude. 4. In contrast, the data are not consistent with the I-II loop and/or the C-terminal forming a unique site for G protein modulation.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Neurônios/fisiologia , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Bovinos , Linhagem Celular , Estimulação Elétrica , Eletrofisiologia , Proteínas de Ligação ao GTP/genética , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Oócitos/metabolismo , Técnicas de Patch-Clamp , Coelhos , Ratos , Transfecção , Xenopus laevisRESUMO
We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.
Assuntos
Canais de Cálcio Tipo N , Canais de Cálcio/genética , Proteínas de Ligação ao GTP/metabolismo , Neurônios/química , Animais , Química Encefálica/fisiologia , Células COS , Canais de Cálcio/química , Canais de Cálcio Tipo R , Proteínas de Transporte de Cátions , Bovinos , Agonistas de Dopamina/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Expressão Gênica/fisiologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Isomerismo , Cinética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estrutura Terciária de Proteína , Quimpirol/farmacologia , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Tionucleotídeos/farmacologiaRESUMO
Tacrine and physostigmine were tested for direct nicotinic actions on Xenopus oocytes microinjected with Torpedo electroplaque membranes. In this preparation, responses to acetylcholine arise 6-8 h after microinjection, due to the incorporation of nicotinic receptors into the plasma membrane by a process not involving protein synthesis. Currents elicited by acetylcholine (100-1000 microM) were recorded by two-electrode voltage clamping. Tacrine (1-1000 microM) and physostigmine (1-100 microM) exerted a potent, reversible block of the nicotinic receptors. The concentration-dependence curves fitted simple hyperbolas, suggesting a stoichiometry of 1:1 in the drug-channel interactions. Currents elicited by the highest acetylcholine concentration were inhibited by tacrine with maximal affinity, indicating an action at a site other than the ligand-binding domain. Inhibition was reduced at depolarising potentials, which is consistent with a preferential interaction with the ligand-bound form of the receptor. Blockade by tacrine or physostigmine was accompanied by a concentration-dependent slowing of the desensitisation, resembling the action of local anaesthetics. These results could indicate a modulatory effect of these drugs on neurosecretion through nicotinic receptors.
Assuntos
Inibidores da Colinesterase/farmacologia , Antagonistas Nicotínicos/farmacologia , Fisostigmina/farmacologia , Receptores Nicotínicos/metabolismo , Tacrina/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Oócitos/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Torpedo , XenopusRESUMO
1. The role of ATP, which is co-released with acetylcholine in synaptic contacts of Torpedo electric organ, was investigated by use of suramin. Suramin [8-(3-benzamido-4-methylbenzamido)naphthalene-1,3,5-trisulphoni c acid], a P2 purinoceptor antagonist, potently inhibited in a non-competitive manner the ecto-apyrase activity associated with plasma membrane isolated from cholinergic nerve terminals of Torpedo electric organ. The Ki was 30 microM and 43 microM for Ca(2+)-ADPase and Ca(2+)-ATPase respectively. 2. In Torpedo electric organ, repetitive stimulation decreased the evoked synaptic current by 51%. However, when fragments of electric organ were incubated with suramin the evoked synaptic current declined by only 14%. Fragments incubated with the selective A1 purinoceptor antagonist, DPCPX, showed 5% synaptic depression. 3. The effects of suramin and DPCPX on synaptic depression were not addictive. Synaptic depression may thus be linked to endogenous adenosine formed by dephosphorylation of released ATP by an ecto-apyrase. The final effector in synaptic depression, adenosine, acts via the A1 purinoceptor. 4. ATP hydrolysis is prevented in the presence of suramin. It slightly increased (20%) the mean amplitude of spontaneous miniature endplate currents. The frequency distribution of the amplitude of spontaneous events was shifted to the right, indicating that ATP, when not degraded, may modulate the activation of nicotinic acetylcholine receptors activated by the quantal secretion of acetycholine.
Assuntos
Trifosfato de Adenosina/farmacologia , Apirase/antagonistas & inibidores , Suramina/farmacologia , Acetilcolina/fisiologia , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Apirase/metabolismo , Membrana Celular/fisiologia , Órgão Elétrico/efeitos dos fármacos , Órgão Elétrico/enzimologia , Órgão Elétrico/metabolismo , Eletrofisiologia , Potenciais da Membrana/efeitos dos fármacos , Placa Motora/efeitos dos fármacos , Terminações Nervosas/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Receptores Colinérgicos/fisiologia , Transmissão Sináptica , Torpedo , Xantinas/farmacologiaRESUMO
We have developed a new method for the generation of functionally active presynaptic chimeras in Xenopus laevis oocytes. Frog oocytes injected with presynaptic subcellular fractions extracted from the electric organ of Torpedo marmorata release acetylcholine in a calcium-dependent manner upon chemical stimulation. Neither oocytes injected without presynaptic plasma membranes nor oocytes injected with ghost erythrocyte plasma membrane instead of presynaptic plasma membrane release acetylcholine. This suggests that specific presynaptic components necessary for KCl-evoked, Ca(2+)-dependent acetylcholine release become functionally integrated in the Xenopus laevis oocytes. Moreover, rhodaminated presynaptic plasma membranes and the synaptic vesicle protein synaptophysin are detected on the oocyte surface by fluorescence or immunofluorescence, respectively, showing that the injected presynaptic components are incorporated into the membrane of the frog oocyte. Furthermore, Botulinum neurotoxin type A, a specific blocker of acetylcholine release in the neuromuscular junction, inhibits the neurotransmitter release from the chimerical oocytes. This suggests that targets for toxin action are also functionally incorporated in the oocyte upon injection of membranous presynaptic components. Our results show that oocytes injected with presynaptic components behave as cholinergic nerve ending chimeras, at least in terms of neurotransmitter release and toxin targets. The system bypasses some problems associated with messenger RNA expression because not only proteins, but native presynaptic components are incorporated. This new technique may provide a useful approach for electrophysiological and pharmacological studies in order to characterize the synaptic transmission.
Assuntos
Acetilcolina/metabolismo , Toxinas Botulínicas/toxicidade , Cálcio/farmacologia , Oócitos/fisiologia , Cloreto de Potássio/farmacologia , Membranas Sinápticas/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Quimera , Feminino , Imuno-Histoquímica , Fusão de Membrana , Camundongos , Microinjeções , Neurotoxinas/toxicidade , Oócitos/efeitos dos fármacos , RNA Mensageiro/biossíntese , Sinaptofisina/análise , Extratos de Tecidos/farmacologia , Torpedo , Xenopus laevisRESUMO
1. The anticholinesterases, tacrine (100 microM) and physostigmine (60 microM) had different effects on the amplitude distribution and kinetics of miniature endplate currents (m.e.p.cs) recorded extracellularly from the electric organ of Torpedo marmorata. 2. Tacrine increased the ratio of giant miniatures (larger than 4 mV of amplitude) to more than 20% of recorded spontaneous events. In the presence of physostigmine such events represented only 4%. 3. Both tacrine and physostigmine increased the rise time and the decay phase of normal-sized m.e.p.cs when compared to control conditions. Both effects were significantly greater for tacrine. 4. We have tested the specificity of the tacrine effect on ectoenzyme activities associated with plasma membranes of these pure cholinergic nerve endings. Tacrine does not act unspecifically on every ectoenzyme, because it is not able to block the ectoapyrase activity even at a concentration 100 fold greater than that required to inhibit 94% of AChE. 5. We conclude that the differential effects of tacrine and physostigmine can be explained in terms of undetermined presynaptic actions of tacrine, while comparable effects of the two compounds can be explained through a shared anticholinesterase activity.