RESUMO
The mechanism of mediator secretion from mast cells in disease is likely to include modulation of ion channel activity. Several distinct Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no data on human mast cells. We have used the whole-cell variant of the patch clamp technique to characterize for the first time macroscopic ion currents in purified human lung mast cells and human peripheral blood-derived mast cells at rest and following IgE-dependent activation. The majority of both mast cell types were electrically silent at rest with a resting membrane potential of around 0 mV. Following IgE-dependent activation, >90% of human peripheral blood-derived mast cells responded within 2 min with the development of a Ca(2+)-activated K(+) current exhibiting weak inward rectification, which polarized the cells to around -40 mV and a smaller outwardly rectifying Ca(2+)-independent Cl(-) conductance. Human lung mast cells showed more heterogeneity in their response to anti-IgE, with Ca(2+)-activated K(+) currents and Ca(2+)-independent Cl(-) currents developing in approximately 50% of cells. In both cell types, the K(+) current was blocked reversibly by charybdotoxin, which along with its electrophysiological properties suggests it is carried by a channel similar to the intermediate conductance Ca(2+)-activated K(+) channel. Charybdotoxin did not consistently attenuate histamine or leukotriene C(4) release, indicating that the Ca(2+)-activated K(+) current may enhance, but is not essential for, the release of these mediators.
Assuntos
Canais Iônicos/fisiologia , Mastócitos/imunologia , Canais de Potássio Cálcio-Ativados/fisiologia , Adulto , Células Sanguíneas/imunologia , Degranulação Celular , Condutividade Elétrica , Sangue Fetal , Liberação de Histamina , Humanos , Imunoglobulina E , Ativação do Canal Iônico , Leucotrieno C4/metabolismo , Pulmão/citologia , Pulmão/imunologia , Técnicas de Patch-ClampRESUMO
The mechanisms underlying the recruitment, differentiation, and sustained activation of mast cells in disease are likely to include modulation of ion channels. Specific Ca(2+), K(+), and Cl(-) conductances have been identified in rodent mast cells, but there are no equivalent data on human mast cells. We have used the whole-cell patch-clamp technique to characterize macroscopic ion currents in both the human mast cell line HMC-1 and human skin mast cells (HSMCs) at rest and in HMC-1 after activation with calcium ionophore. HSMCs were electrically silent at rest. In contrast, HMC-1 expressed a strong outwardly rectifying voltage-dependent Cl(-) conductance characteristic of ClC-4 or ClC-5 and a small inwardly rectifying K(+) current not carried by the classical Kir family of K(+) channels. Calcium ionophore induced the appearance of outwardly rectifying Ca(2+)-activated Cl(-) and K(+) currents, while hypotonicity induced another outwardly rectifying conductance typical of ClC-3. Reverse transcription-PCRs confirmed that mRNAs for the voltage-dependent Cl(-) channels ClC-3 and -5 were expressed. This is the first definitive description of a ClC-4/5-like current in a native leukocyte. We suggest that this current may contribute to the malignant phenotype while the Ca(2+)-activated K(+) and Cl(-) currents may be involved in cell activation.
Assuntos
Cálcio/farmacologia , Canais Iônicos/fisiologia , Mastócitos/fisiologia , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Leucemia de Mastócitos , Mastócitos/química , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Pele/citologia , Células Tumorais CultivadasRESUMO
Efflux of intracellular K(+) and cell shrinkage are features of apoptosis in many experimental systems, and a regulatory role has been proposed for cytoplasmic [K(+)] in initiating apoptosis. We have investigated this in both death-receptor-mediated and chemical-induced apoptosis. Using Jurkat T cells pre-loaded with the K(+) ion surrogate (86)Rb(+), we have demonstrated an efflux of intracellular K(+) during apoptosis that was concomitant with, but did not precede, other apoptotic changes, including phosphatidylserine externalization, mitochondrial depolarization and cell shrinkage. To further clarify the role of K(+) ions in apoptosis, cytoprotection by elevated extracellular [K(+)] was studied. Induction of apoptosis by diverse death-receptor and chemical stimuli in two cell lines was inhibited prior to phosphatidylserine externalization, mitochondrial depolarization, cytochrome c release and caspase activation. Using a cell-free system, we have demonstrated a novel mechanism by which increasing [K(+)] inhibited caspase activation. In control dATP-activated lysates, Apaf-1 oligomerized to a biologically active caspase processing approximately 700 kDa complex and an inactive approximately 1.4 MDa complex. Increasing [K(+)] inhibited caspase activation by preventing formation of the approximately 700 kDa complex, but not of the inactive complex. Thus intracellular and extracellular [K(+)] markedly affect caspase activation and the initiation of apoptosis induced by both death-receptor ligation and chemical stress.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Potássio/farmacologia , Receptores do Fator de Necrose Tumoral/fisiologia , Linfócitos T/citologia , Linfócitos T/fisiologia , Clorometilcetonas de Aminoácidos/farmacologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Citosol/fisiologia , Ativação Enzimática , Etoposídeo/farmacologia , Espaço Extracelular/fisiologia , Citometria de Fluxo , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/fisiologia , Células Jurkat , Cinética , Potenciais da Membrana/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Fosfatidilserinas/metabolismo , Cloreto de Potássio/farmacologia , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Rubídio/farmacocinética , Linfócitos T/efeitos dos fármacos , Células Tumorais Cultivadas , Receptor fas/efeitos dos fármacos , Receptor fas/fisiologiaRESUMO
P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.
Assuntos
Infertilidade Masculina/etiologia , Receptores Purinérgicos P2/fisiologia , Ducto Deferente/fisiopatologia , Animais , Anticoncepção , Ejaculação/fisiologia , Feminino , Deleção de Genes , Infertilidade Masculina/genética , Masculino , Camundongos , Contração Muscular , Músculo Liso/fisiologia , Gravidez , Agonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Reprodução , Contagem de Espermatozoides , Sistema Nervoso Simpático/fisiologiaRESUMO
Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.
Assuntos
Cerebelo/metabolismo , Hipocampo/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Epêndima/metabolismo , Imuno-Histoquímica , Masculino , Bulbo/metabolismo , Músculo Liso Vascular/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Canais de Potássio/genética , Canais de Potássio/imunologia , Coelhos , Ratos , Ratos Endogâmicos F344 , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Distribuição TecidualAssuntos
Cerebelo/citologia , Locus Cerúleo/citologia , Neurônios/citologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/análise , Receptores da Neurocinina-1/análise , Animais , Células CHO , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Cricetinae , Imuno-Histoquímica , Oligodendroglia/citologia , Ratos , Proteínas Recombinantes/análise , TransfecçãoRESUMO
Externally applied Ag+ (100-200 nM) irreversibly blocked the strong inwardly rectifying K+ channel, Kir2.1. Mutation to serine of a cysteine residue at position 149 in the pore-forming H5 region of Kir2.1 abolished Ag+ blockage. To determine how many of the binding sites must be occupied by Ag+ before the channel is blocked, we measured the rate of channel block and found that our results were best fitted assuming that only one Ag+ ion need bind to eliminate channel current. We tested our hypothesis further by constructing covalently linked dimers and tetramers of Kir2.1 in which cysteine had been replaced by serine in one (dimer) or three (tetramer) of the linked subunits. When expressed, these constructs yielded functional channels with either two (dimer) or one (tetramer) cysteines per channel at position 149. Blockage in the tetramer was complete after sufficient exposure to 200 nM Ag+, a result that is also consistent with only one Ag+ being required to bind to Cys149 to block fully. The rate of development of blockage was 16 times slower than in wild-type channels; the rate was 4 times slower in channels formed from dimers.
Assuntos
Cisteína , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/química , Canais de Potássio/fisiologia , Prata/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Sequência Conservada , Cricetinae , Dimerização , Cinética , Substâncias Macromoleculares , Camundongos , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Bloqueadores dos Canais de Potássio , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , TransfecçãoAssuntos
Sistema Cardiovascular/metabolismo , GMP Cíclico/metabolismo , Canais Iônicos/genética , Sequência de Aminoácidos , Animais , Bovinos , Canais de Cátion Regulados por Nucleotídeos Cíclicos , DNA Complementar/genética , Expressão Gênica , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Células Fotorreceptoras/metabolismo , Sistemas do Segundo Mensageiro , Suínos , Distribuição TecidualRESUMO
1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane-spanning alpha-helix of the murine inward rectifier, IRK1, by site-directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.
Assuntos
Ácido Aspártico , Magnésio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Asparagina , Sequência de Bases , Sítios de Ligação , Membrana Celular/fisiologia , Sequência Conservada , Ácido Glutâmico , Ativação do Canal Iônico , Leucemia Eritroblástica Aguda , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Mutação Puntual , Canais de Potássio/biossíntese , Canais de Potássio/efeitos dos fármacos , Estrutura Secundária de Proteína , Transfecção , Células Tumorais CultivadasRESUMO
We have measured the expression levels of a range of distinct ion channel genes in the apex/ventricle region and sino-atrial node (SAN) sub-regions of heart under conditions in which conventional Northern hybridization or ribonuclease protection methods were too insensitive or non-quantitative. The abundance of six potassium channel mRNAs was determined in relation to a single synthetic competitor RNA template which was co-reverse transcribed and PCR-amplified. By these methods we have shown that coronary artery ligation procedures which induce anoxia and ischaemic scarring in the apical region reduce amplifiable message abundance in a time-dependent, but non-specific manner. There was no evidence for any selective reduction of individual mRNA levels during this process. Despite a high reproducibility of titration endpoints, competitive RNA template amplification assays did not provide a simple marker for ischaemic damage, since it was not possible to control for tissue sample heterogeneity. We have also applied these competitive nucleic acid titration techniques to demonstrate expression of cAMP- and cGMP-gated ion channel sequences in small pieces of tissue derived from the SAN sub-region of rabbit heart. Although the ion channels encoded by these sequences are obligately coupled to intracellular signalling agonists commonly found in cardiac cells, they have not been described in functional terms within SAN or any other cardiac subregion. For rapid determination of cDNA molecular numbers, we have devised single-gene, DNA template controls to measure absolute abundance of a cAMP-gated cDNA derived from heart tissue. Competitive titration procedures therefore provide an important technique for probing gene induction and/or repression accompanying pharmacological or surgical interventions, or in progression of disease states. For rare cDNAs, they can estimate the representativeness of a given preparation prior to library construction, screening and retrieval of clones, while eliminating 'false positive' or 'false negative' signals.
Assuntos
Canais Iônicos/genética , Isquemia Miocárdica/genética , Miocárdio/química , RNA Mensageiro/análise , RNA Mensageiro/genética , Animais , Sequência de Bases , Northern Blotting , Primers do DNA , Amplificação de Genes , Ventrículos do Coração/química , Ventrículos do Coração/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Canais Iônicos/fisiologia , Masculino , Dados de Sequência Molecular , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Reação em Cadeia da Polimerase , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio/fisiologia , RNA Mensageiro/metabolismo , Coelhos , Ratos , Ratos Sprague-Dawley , Nó Sinoatrial/química , Nó Sinoatrial/metabolismo , Titulometria , Transcrição GênicaRESUMO
1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole-cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e-fold as the driving force on K+(V-EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current-voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.
Assuntos
Ativação do Canal Iônico/fisiologia , Magnésio/fisiologia , Canais de Potássio/metabolismo , Potássio/fisiologia , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , Eletrofisiologia , Genoma , Íntrons , Leucemia Eritroblástica Aguda/metabolismo , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Canais de Potássio/efeitos dos fármacosAssuntos
Proteínas do Olho/genética , Canais Iônicos , Nó Sinoatrial/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Humanos , Camundongos , Dados de Sequência Molecular , Coelhos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Fundamental studies of the mechanism of human beta-like globin gene-expression have identified DNA sequences (locus control regions or LCRs) which directly influence the specific activation of beta-globin genes in erythroid cells. Here we report the first applications of LCR-mediated gene-activation to stable electrophysiological expression of several homomultimeric ion channel proteins. We describe expression driven from a native K+ channel gene promoter region, contiguous to an intronless coding sequence, within a single excised human genomic DNA fragment. In addition, cDNAs encoding both inactivating and non-inactivating mammalian K+ currents have been expressed by insertion between the promoter and second intron of the human beta-globin gene. Expression levels are characteristically independent of integration position and are proportional to LCR-gene copy number, a parameter specific for each clonal cell line. K+ channel expression is inducible by erythroid differentiation and has been demonstrated by electrophysiological recordings of cells taken directly from culture.
Assuntos
Regulação da Expressão Gênica , Globinas/genética , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Eletrofisiologia , Células Precursoras Eritroides/metabolismo , Ligação Genética , Humanos , Potenciais da Membrana , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Ativação TranscricionalAssuntos
Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Glibureto/metabolismo , Ilhotas Pancreáticas/fisiologia , Canais de Potássio/fisiologia , Compostos de Sulfonilureia/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Membrana Celular/fisiologia , Modelos Biológicos , Canais de Potássio/efeitos dos fármacosRESUMO
Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.
Assuntos
DNA Viral/metabolismo , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Plasmídeos , Recombinases Rec A/metabolismo , Bacteriófago phi X 174/metabolismo , DNA Circular/metabolismo , DNA de Cadeia Simples/metabolismo , Cinética , Substâncias Macromoleculares , Modelos Estruturais , Ligação ProteicaRESUMO
The RecA protein from E. coli gains access to duplex DNA, by nucleation from a short single-stranded gap, to form a spiral nucleoprotein filament that is capable of interaction with homologous duplex DNA. The observations described here demonstrate that any part of the nucleoprotein filament, whether it contains single- or double-stranded DNA, is capable of pairing with homologous duplex DNA. Homologous contacts between regions of duplex DNA lead to an increase in the initial rate and final extent of joint molecule formation. The experiments indicate that pairing is facilitated by the formation of nascent synaptic intermediates between duplex DNA sequences. Using chimeric form I DNA, which is incapable of forming an inter-wound or plectonemic joint with the gapped DNA due to the presence of flanking heterologous sequences, we show that these duplex-duplex pairing reactions involve extensive underwinding of the double helix.