RESUMO
The olfactory receptor (OR) family was found to be expressed mainly in the nasal epithelium. In the last two decades members of the OR family were detected to be functional expressed in different parts of the human body such as in liver, prostate or intestine cancer cells. Here, we detected the expression of several ORs in the human chronic myelogenous leukemia (CML) cell line K562 and in white blood cells of clinically diagnosed acute myeloid leukemia (AML) patients by RT-PCR and next-generation sequencing. With calcium-imaging, we characterized in greater detail the cell biological role of one OR (OR2AT4) in leukemia. In both cell systems, the OR2AT4 agonist Sandalore-evoked strong Ca(2+) influx via the adenylate cyclase-cAMP-mediated pathway. The OR2AT4 antagonist Phenirat prevented the Sandalore-induced intracellular Ca(2+) increase. Western blot and flow cytometric experiments revealed that stimulation of OR2AT4 reduced the proliferation by decreasing p38-MAPK phosphorylation and induced apoptosis via phosphorylation of p44/42-MAPK. Furthermore, Sandalore increased the number of hemoglobin-containing cells in culture. We described for the first time an OR-mediated pathway in CML and AML that can regulate proliferation, apoptosis and differentiation after activation. This mechanism offers novel therapeutic options for the treatment of AML.
RESUMO
The ectopic expression of olfactory receptors (ORs) in the human body has been of major interest in the past decade. Several studies have reported the expression of ORs not only in healthy tissues such as heart, sperm or skin cells, but also in cancerous tissues of the liver, prostate or intestine. In the present study, we detected the expression of OR51B5 in the chronic myelogenous leukemia (CML) cell line K562 and in white blood cell samples of clinically diagnosed acute myelogenous leukemia (AML) patients by reverse transcription-PCR and immunocytochemical staining. The known OR51B5 ligand isononyl alcohol increased the levels of intracellular Ca(2+) in both AML patient blood cells and K562 cells. With calcium imaging experiments, we characterized in greater detail the OR51B5-mediated signaling pathway. Here, we observed an involvement of adenylate cyclase and the downstream L-type and T-type calcium channels. In addition, the activation of OR51B5 leads to an inhibition of cell proliferation in K562 cells. In western blot experiments, we found that incubation with isononyl alcohol led to a reduction in p38-MAPK (mitogen-activated protein kinase) phosphorylation that might be responsible for the decreased cell proliferation. In the present study, we characterized the OR51B5-mediated signaling pathway downstream of the activation with isononyl alcohol, which leads to reduced proliferation and therefore provide a novel pharmacological target for CML and AML, the latter of which remains difficult to treat.
RESUMO
Specific anosmia, the inability to perceive a specific odor, while olfactory perception is otherwise intact, is known as a rather seldom phenomenon. By testing the prevalence of specific anosmia to 20 different odors in a sample of 1600 people, we were able to estimate the general prevalence of anosmia. This revealed that specific anosmia is not rare at all. In contrast, the general likelihood for specific anosmia approaches 1. In addition, specific anosmia can be very well reversed by "smell training" during the course of 3 months. To summarize, specific anosmia seems to be a rule, not an exception, of olfactory sensation. The lack of perception of certain odors may constitute a flexible peripheral filter mechanism, which can be adapted by exposure to odors.
Assuntos
Encéfalo/fisiopatologia , Odorantes , Transtornos do Olfato/epidemiologia , Transtornos do Olfato/fisiopatologia , Percepção Olfatória , Limiar Sensorial , Alemanha/epidemiologia , Humanos , Transtornos do Olfato/prevenção & controle , PrevalênciaRESUMO
BACKGROUND AND PURPOSE: Transient receptor potential (TRP) V3 is a thermosensitive ion channel expressed predominantly in the skin and neural tissues. It is activated by warmth and the monoterpene camphor and has been hypothesized to be involved in skin sensitization. A selection of monoterpenoid compounds was tested for TRPV3 activation to establish a structure-function relationship. The related channel TRPM8 is activated by cool temperatures and a number of chemicals, among them the monoterpene (-)-menthol. The overlap of the receptor pharmacology between the two channels was investigated. EXPERIMENTAL APPROACH: Transfected HEK293 cells were superfused with the test substances. Evoked currents were measured in whole cell patch clamp measurements. Dose-response curves for the most potent agonists were obtained in Xenopus laevis oocytes. KEY RESULTS: Six monoterpenes significantly more potent than camphor were identified: 6-tert-butyl-m-cresol, carvacrol, dihydrocarveol, thymol, carveol and (+)-borneol. Their EC(50) is up to 16 times lower than that of camphor. All of these compounds carry a ring-located hydroxyl group and neither activates TRPM8 to a major extent. CONCLUSIONS AND IMPLICATIONS: Terpenoids have long been recognized as medically and pharmacologically active compounds, although their molecular targets have only partially been identified. TRPV3 activation may be responsible for several of the described effects of terpenoids. We show here that TRPV3 is activated by a number of monoterpenes and that a secondary hydroxyl-group is a structural requirement.
Assuntos
Monoterpenos/farmacologia , Canais de Cátion TRPV/agonistas , Animais , Células Cultivadas , Desenho de Fármacos , Feminino , Humanos , Relação Estrutura-Atividade , Canais de Cátion TRPM/agonistas , Xenopus laevisRESUMO
Glycine receptors are ligand-gated ion channel proteins mediating synaptic inhibition in the spinal cord, retina and brain of vertebrates. We have cloned and functionally characterized two glycine receptor alpha-subunits from the perch (Roccus americana) retina. Based on sequence homology with the mammalian counterparts, we termed these subunits alpha 1 and alpha 3. RT-PCR revealed the presence of both subunits in retina and brain, whereas alpha1 was predominant in spinal cord. A short splice variant of alpha1 was detected in the brain but not in the retina. Functional expression of the perch subunits in HEK-293 cells yielded robust glycine-gated currents sensitive to strychnine. The perch receptors displayed a high efficacy for taurine and GABA and thus differ from the mammalian counterparts. Because the retina is a rich source for taurine, this finding could be of physiological importance. The structural features of the ligand binding domain strongly support the notion of increased glycine/GABA discrimination in higher vertebrates.
Assuntos
Percas , Receptores de Glicina/fisiologia , Retina/química , Sequência de Aminoácidos , Animais , Química Encefálica , Clonagem Molecular , Eletrofisiologia , Antagonistas GABAérgicos/farmacologia , Glicina/farmacocinética , Glicinérgicos/farmacologia , Dados de Sequência Molecular , Picrotoxina/farmacologia , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/química , Estricnina/farmacologia , Taurina/farmacocinética , Ácido gama-Aminobutírico/farmacocinéticaRESUMO
The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants starts with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have expressed and characterized different olfactory receptors with several expression systems. Here we provide the first documentation of functional expression of odorant receptors using the Semliki Forest virus system. The human odorant OR 17-40 receptor and the rat 17 receptor were functionally expressed in vertebrate kidney cells (HEK293) using recombinant Semliki Forest viruses. Receptors were expressed as a fusion protein with the N-terminal membrane import sequence of the guinea pig serotonin receptor. Experiments employing the Ca2+-sensitive dye fura-2 revealed a fast, transient increase in the [Ca2+]i after application of the specific agonists helional and octanal to HEK293 cells infected with viruses containing RNA for the human odorant OR 17-40 receptor and the rat 17 receptor, respectively.
Assuntos
Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Vírus da Floresta de Semliki/genética , Animais , Cálcio/metabolismo , Células Cultivadas , Clonagem Molecular , Corantes Fluorescentes/análise , Fura-2/análise , Humanos , Imuno-Histoquímica , Octanóis/farmacologia , Fenobarbital/farmacologia , Engenharia de Proteínas/métodos , Ratos , Receptores Odorantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Odorant receptors (ORs) constitute the molecular basis for the detection of volatile odorous molecules and the perception of smell. Our understanding of chemical senses has been greatly expanded by the discovery of the OR gene families in vertebrates and in the nematode Caenorhabditis elegans. Recently, candidate Drosophila OR genes have been identified. The putative ORs do not possess any primary sequence identity with known vertebrate or C. elegans receptors, but belong to the family of G protein-coupled receptors according to their predicted seven transmembrane topology. To prove olfactory function of these proteins, we expressed a member of the putative Drosophila OR gene family, Or43a, in Xenopus laevis oocytes. Using two-electrode voltage-clamp recording we identified four odors (cyclohexanone, cyclohexanol, benzaldehyde, and benzyl alcohol) that activated the receptor at low micromolar concentration and structurally related substances that did not. This report shows the function and specificity of a member of the recently identified family of Drosophila ORs expressed in a heterologous system.
Assuntos
Drosophila/metabolismo , Receptores Odorantes/metabolismo , Animais , Sequência de Bases , Primers do DNA , DNA Complementar , Feminino , Potenciais da Membrana , Técnicas de Patch-Clamp , Receptores Odorantes/efeitos dos fármacos , Receptores Odorantes/genética , Transdução de Sinais , Xenopus laevisRESUMO
Desensitization of ligand-gated ion channels shapes synaptic responses and provides critical neuroprotection at central synapses, yet the molecular mechanisms underlying the desensitization process are poorly understood. Using the whole-cell voltage-clamp technique, we investigated desensitization kinetics of recombinant human and guinea pig alpha-homomeric 5-hydroxytryptamine type 3 (5-HT(3A)) receptors heterologously expressed in human embryonic kidney 293 cells. Human 5-HT(3A) receptors desensitize 3.5 times faster than does the homologous receptor from guinea pigs. By constructing various chimeras and through site-directed mutagenesis, we have identified a single serine in the M1 region of the human 5-HT(3A) receptor sequence (S248) that, when substituted with threonine found in the equivalent guinea pig sequence (T254), conferred guinea pig-like kinetics on the time course of desensitization of the human receptor. Correspondingly, the reverse mutation (guinea pig T254S) resulted in a fast, human-like time constant of desensitization. Thus, the primary structure of the M1 region is an important determinant of desensitization kinetics of recombinant 5-HT(3A) receptors.
Assuntos
Rim/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Cobaias , Humanos , Rim/citologia , Cinética , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína/fisiologia , Receptores 5-HT3 de Serotonina , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serotonina/metabolismo , Serotonina/farmacologia , Relação Estrutura-AtividadeRESUMO
The current (I(p)) generated by the wild-type or the glutamate (E) 779 alanine (A) mutant of the rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells was studied at 35 degrees C by means of whole-cell recording in Na(+)-free and Na(+)-containing solution. Glutamate 779 is located in the fifth transmembrane domain of the alpha-subunit of the Na(+)/K(+)-ATPase. Compared with the wild-type, the E779A mutant exhibited an apparent K(+)(o)-affinity decreased by a factor of 3-4 both in Na(+)-free and in Na(+)-containing media. The competition of Na(+)(o) and K(+)(o) for cation binding sites of the pump remained unchanged. Similarly, in Na(+)-free solution the shape of the I(p)-V curves for various external K(+)-concentrations ([K(+)](o)) was essentially the same. However, in Na(+)-containing solutions the shape of I(p)-V curves from cells expressing the mutant of the rat alpha1-subunit clearly differed from the shape observed in cells expressing the wild-type, but voltage dependence of the pump current persisted. A prominent Na(+)(o)-activated, electrogenic Na(+)-transport mediated by the pump, displaying little voltage dependence in the potential range tested (-80 to +60 mV), was present in the cells expressing the E779A mutant pump. The data suggest that exchanging E779 for A in the rat Na(+)/K(+) pump alpha1-subunit causes a modest decrease in the apparent K(+)(o) affinity and a profound, Na(+)(o)-dependent alteration in the electrogenicity of the mutant pump expressed in HEK 293 cells.
Assuntos
ATPase Trocadora de Sódio-Potássio/genética , Animais , Sítios de Ligação , Encéfalo/enzimologia , Linhagem Celular , Meios de Cultura/química , Digitoxigenina , Eletroquímica , Íons/química , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos , Potássio/química , Ratos , Sódio/química , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/química , Soluções , TransfecçãoRESUMO
The 5-hydroxytrptamine3 (5-HT3) receptor is a pentameric complex belonging to the family of ligand-gated ion channels. A variety of studies have suggested that phosphorylation regulates the rate of desensitisation and the size of amplitude of the receptor current. In this study we have examined the phosphorylation of the myc-tagged wild-type 5-HT3A receptor subunits from guinea-pig expressed in HEK293 cells (human embryonic kidney). Stably transfected cells were metabolically labelled with 32P-phosphoric acid. The results of immunoprecipitation and autoradiography demonstrate that both splicc variants of the 5-HT3A receptor subunit are phosphorylated in HEK293 cells. Site-specific mutagenesis revealed that phosphorylation occurs at serine 409, a potential target of protein kinase A. Thus the 5-HT3 receptor might be modulated by intracellular pathways, that allow variable 5-hydroxytryptamine action as responses to different extracellular stimuli.
Assuntos
Receptores de Serotonina/metabolismo , Animais , Autorradiografia , Sequência de Bases , Western Blotting , Linhagem Celular , Primers do DNA , Cobaias , Humanos , Mutagênese Sítio-Dirigida , Fosforilação , Testes de Precipitina , Receptores de Serotonina/genética , Receptores 5-HT3 de Serotonina , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Here, we provide the first evidence for functional expression of a human olfactory receptor protein (OR17-40) and show that recombinant olfactory receptors can be functionally expressed in heterologous systems. A mixture of 100 different odorants (Henkel 100) elicited a transient increase in intracellular [Ca(2+)] in human embryonic kidney 293 (HEK293) cells stably or transiently transfected with the plasmid pOR17-40. By subdividing the odorant mixture into progressively smaller groups, we identified a single component that represented the only effective substance: helional. Only the structurally closely related molecule heliotroplyacetone also activated the receptor. Other compounds, including piperonal, safrole, and vanillin, were completely ineffective. Mock-transfected cells and cells transfected with other receptors showed no change in intracellular [Ca(2+)] in response to odor stimulation. We were also able to functionally express OR17-40 in Xenopus laevis oocytes. Coexpression of a "reporter" channel allowed measurement of the response of oocytes injected with the cRNA of the human receptor to the odor mixture Henkel 100. The effective substances were the same (helional, heliotropylacetone) as those identified by functionally expressing the receptor in HEK293 cells and were active at the same, lower micromolar concentration. These findings open the possibility of now characterizing the sensitivity and specificity of many, if not all, of the hundreds of different human olfactory receptors.
Assuntos
Receptores Odorantes/fisiologia , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/fisiologia , DNA Complementar , Feminino , Humanos , Rim , Potenciais da Membrana/efeitos dos fármacos , Modelos Neurológicos , Dados de Sequência Molecular , Odorantes , Oócitos/fisiologia , Estrutura Secundária de Proteína , RNA Complementar/metabolismo , Receptores Odorantes/química , Receptores Odorantes/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Transfecção , Xenopus laevisRESUMO
The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants begins with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have cloned, functionally expressed and characterized the first human olfactory receptor (OR 17-40). Application of a mixture of hundred different odorants elicited a transient increase in intracellular calcium at HEK 293-cells which were transfected with a plasmid containing the receptor encoding DNA and a membrane import sequence. By subdividing the odorant mixture in smaller groups we could identify a single component which represented the only effective substance: helional. Testing some structurally closely related molecules we found only one other compound which also could activate the receptor: heliotropyl acetone. All other compounds tested were completely ineffective. These findings represent the beginning of molecular understanding of odorant recognition in humans.
Assuntos
Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Benzaldeídos/química , Benzaldeídos/metabolismo , Benzaldeídos/farmacologia , Benzodioxóis , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , Estrenos/farmacologia , Humanos , Dados de Sequência Molecular , Odorantes , Inibidores de Fosfodiesterase/farmacologia , Pirrolidinonas/farmacologia , Receptores Odorantes/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Safrol/química , Safrol/metabolismo , Safrol/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade , TransfecçãoRESUMO
The amino acid sequence BCNG-1 (brain cyclic nucleotide gated 1, of the mouse), the first member of mamalian I(h) channels, was used to construct a set of polymerase chain reaction (PCR) primers from possibly conserved regions. Reverse transcription-PCR with Drosophila melanogaster mRNA yielded in a PCR product, which exhibited a high homology to BCNG-1. Using these PCR products to screen a D. melanogaster head cDNA library we isolated a cDNA encoding a member of a new class of putative voltage- and cyclic nucleotide-gated potassium channels from D. melanogaster. The most important features of the amino acid sequence predicted from the cDNA were a C-terminal cyclic nucleotide-binding region, an S4-voltage sensor and a putative potassium-selective pore-forming motif. The high homology of 51% to the sea urchin I(h) channel, which belongs to the same class of ion channels as BCNG-1, leads us to suggest that the Drosophila cDNA is the first insect member of a new class of hyperpolarization-activated and cyclic nucleotide-gated channels. As shown by in situ hybridization, a pronounced mRNA expression was detected in neuronal tissue, including sensory tissue like the compound eyes, and the olfactory and the auditory organs.
Assuntos
Canais de Cálcio/genética , DNA Complementar , Proteínas de Drosophila , Drosophila melanogaster/genética , Neurônios Aferentes/metabolismo , Canais de Potássio/genética , Homologia de Sequência do Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Drosophila melanogaster/citologia , Olho/citologia , Olho/metabolismo , Expressão Gênica , Genes de Insetos , Hibridização In Situ , Canais Iônicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Neurônios Aferentes/citologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Polymerase chain reaction and rapid amplification of cDNA ends were used to isolate cDNAs encoding a 5-hydroxytryptamine3 (5-HT3) receptor subunit and its splice variants from guinea pig intestine. The amino acid sequence predicted from this cDNA is 81% homologous to the murine 5-HT3 receptor subunits cloned from NCB20 and N1E-115 cells. The splice variants code for two proteins differing by a deletion of six amino acids located in the large intracellular loop between transmembrane domains M3 and M4. For characterization, the cloned 5-HT3 cDNA was expressed in HEK 293 cells, and the electrophysiological and pharmacological properties of the recombinant ion/channel/receptor complex were investigated by patch clamping. Our data reveal that the cloned cDNAs code for guinea pig 5-HT3 receptors, which functionally assemble as homo-oligomers. The kinetic behavior of the ion channel and its sensitivity to several agonists and antagonists were markedly different from those of the cloned 5-HT3 receptors from mouse and human under similar experimental conditions. The agonists used were 5-hydroxytryptamine, 2-methyl-5-hydroxytryptamine, 1-phenylbiguanide (PBG), m-chlorophenylbiguanide, and the antagonists tropisetron and metoclopramide. In addition, 5-HT, PBG, and tropisetron were investigated through radioligand binding to isolated membranes. Compared with the human and murine 5-HT3 receptors, the guinea pig receptor showed prolonged desensitization kinetics. In addition, the guinea pig 5-HT3 receptor did not respond to the selective 5-HT3 receptor agonist PBG. Construction of chimeric receptors between guinea pig and human 5-HT3 receptor sequences localized the differences in desensitization kinetics to the carboxyl-terminal domain and the ligand binding site to the amino-terminal domain of the receptor protein. Molecular determinants of the PBG binding site of the human 5-HT3 receptor were localized to a 28-amino-acid spanning region adjacent to the M1 region.
Assuntos
Receptores de Serotonina/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Eletrofisiologia , Cobaias , Humanos , Imidazóis/metabolismo , Indóis/metabolismo , Camundongos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , RNA Mensageiro/genética , Receptores de Serotonina/efeitos dos fármacos , Receptores 5-HT3 de Serotonina , Proteínas Recombinantes de Fusão , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Relação Estrutura-Atividade , TransfecçãoRESUMO
Odorant receptors of zebrafish and C elegans were functionally expressed in vertebrate kidney cells (HEK293) using the eucaryotic expression vector pSMyc. Receptor-encoding cDNA cloned into this vector was expressed as a fusion protein with the N-terminal membrane import sequence of the guinea-pig serotonin receptor followed by a myc tag. Immunocytochemical evidence indicates that this strategy directs a protein with the predicted immunoreactivity and approximate molecular weight to the plasma membrane. Fish food extract (TetraMin) evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for three fish odorant receptors and converted to stable cell lines. The effect of the extract was concentration dependent and limited to the fraction of the extract < 5 kDa. Pretreating the transfected cells with the PLC inhibitor U73122 reduced the odor-evoked signal. Fish food extract also evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids containing cDNA for single fish odorant receptors. Diacetyl evoked a transient increase in intracellular [Ca2+] in HEK293 cells transiently transfected with plasmids encoding the cDNA of ODR10, an odorant receptor of C. elegans suggested in other work to be specific for diacetyl. These results strongly imply that odorant receptors can be functionally expressed in HEK293 cells using this novel expression protocol.
Assuntos
Caenorhabditis elegans/genética , Receptores Odorantes/genética , Receptores Odorantes/fisiologia , Peixe-Zebra/genética , Animais , Sequência de Bases , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/fisiologia , Expressão Gênica/genética , Expressão Gênica/fisiologia , Vetores Genéticos , Humanos , Imuno-Histoquímica , Ligantes , Dados de Sequência Molecular , Receptores Odorantes/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/fisiologia , Transfecção/genética , Transfecção/fisiologiaRESUMO
RACE (rapid amplification of cDNA ends) is commonly used for identification and isolation of 3'and 5'termini of cDNA. We developed an improvement of the RACE-method that allows the enrichment of wanted fragments. The important new feature is the purification of the amplified products by biotinylated oligonucleotides that hybridize internally. Hybrids are isolated by streptavidin coated magnetic particles.
Assuntos
Primers do DNA/síntese química , Oligodesoxirribonucleotídeos/síntese química , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismo , Receptores de Serotonina/biossíntese , Sequência de Bases , Biotina , Colo , Primers do DNA/química , DNA Complementar , Biblioteca Gênica , Humanos , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/isolamento & purificação , Receptores 5-HT3 de SerotoninaRESUMO
Subunit composition of subsynaptic transmitter receptors is controlled presynaptically in the developing neuromuscular junction. To investigate presynaptic regulation of NMDA receptor subunit composition in the CNS, we co-cultured different types of hippocampal explants with dissociated target neurons. Postsynaptic NMDA receptors were studied using whole-cell patch-clamp recordings. After 1 week in culture with innervation by dentate gyrus (dg) explants, the kinetic and pharmacological properties of postsynaptic NMDA receptors indicated the expression of NMDA receptor subtypes containing NR2B subunits (NR1/NR2A/NR2B or NR1/NR2B or both). The properties of NMDA receptors in noninnervated neurons were similar to those of neurons innervated by dg explants. In contrast, after innervation by explants from the cornu ammonis (CA) region, we found an additional NMDA receptor subtype with properties consistent with the subunit composition NR1/NR2A. These findings indicate that presynaptic signals determine NMDA receptor subunit composition. After prolonged cultivation (11-12 d) the properties of synaptic NMDA receptors in the majority of dg-innervated neurons also indicated the expression of NR1/NR2A receptors. This suggests a delayed developmental maturation of NMDA receptors in dg-innervated neurons. Long-term plasticity of central glutamatergic synapses is critically influenced by the subunit composition of NMDA receptors, and thus presynaptic control of NMDA receptor subunit composition might regulate synaptic plasticity.
Assuntos
Hipocampo/fisiologia , N-Metilaspartato/farmacologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores de N-Metil-D-Aspartato/biossíntese , Sinapses/fisiologia , Animais , Bicuculina/análogos & derivados , Bicuculina/farmacologia , Células Cultivadas , Técnicas de Cocultura , Embrião de Mamíferos , Potenciais Evocados/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Cinética , Substâncias Macromoleculares , Magnésio/farmacologia , Plasticidade Neuronal , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Reação em Cadeia da Polimerase , Quinoxalinas/farmacologia , Ratos , Receptores de N-Metil-D-Aspartato/químicaRESUMO
The Na/K pump current I(p) of single HEK293 cells either untransfected (endogenous I(p)) or transfected with the alpha1 subunit of the rat Na/K pump (exogenous I(p)) was investigated in Na-containing solution by means of whole-cell recording at 30 degrees C. The endogenous I(p) was irreversibly blocked by 10(-4) M ouabain or 2 x 10(-4) M dihydro-ouabain (DHO). Its density amounted to 0.33 pA pF(-1) at 0 mV and 5.4 mM K(o). It was half maximally activated at 1.5 mM K(o) and increased linearly with depolarization over the entire voltage range studied (-80 to +60 mV). In contrast, HEK293 cells stably transfected with cDNA for the cardiac glycoside-resistant alpha1 subunit of the rat Na/K pump showed an I(p) in the presence of 10(-4) M ouabain and 2 x 10(-4) M DHO, respectively. This exogenous I(p) was reversibly blocked by 10(-2) M ouabain. Half maximal activation of the exogenous I(p) occurred at 1.7 mM K(o). Its amplitude increased linearly with depolarization at negative voltages but remained almost constant at positive membrane potentials. Comparison with the I(p) of isolated rat cardiac ventricular myocytes strongly suggests that the exogenous I(p) in HEK293 cells is generated by the alpha1 subunit of the rat Na/K pump since it displays identical properties. Therefore, HEK293 cells represent an expression system well suited for the electrophysiological analysis of recombinant, cardiac glycoside-resistant Na/K pumps by means of whole-cell recording.
Assuntos
Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/enzimologia , Condutividade Elétrica , Humanos , Miocárdio/metabolismo , Técnicas de Patch-Clamp , Potássio/farmacologia , Ratos , Proteínas Recombinantes/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , TransfecçãoRESUMO
A cDNA with an open reading frame of 1929 bp (termed sir) was isolated from a lambda ZapII library of Arabidopsis thaliana leaf tissue. The polypeptide sequence deduced from the cDNA is homologous to the ferredoxin-dependent sulfite reductase (EC 1.8.7.1) from Synechococcus PCC7942 and distantly related to the hemoprotein subunit of Escherichia coli NADPH-dependent sulfite reductase (EC 1.8.1.2). A molecular mass of 71.98 kDa can be predicted for a ferredoxin sulfite reductase from A. thaliana. The polypeptide consists of 642 amino acids including a transit peptide of 66 residues (6.72 kDa) that is assumed to direct the protein into the plastid. For expression and enzymatic characterization of a putative A. thaliana ferredoxin sulfite reductase, the DNA of the transit peptide was deleted by a PCR method. The truncated cDNA clone was expressed as his-tag fusion protein. The modified gene product was enzymatically inactive but specific cross-reaction with polyclonal antibodies against ferredoxin sulfite reductase from Synechococcus is seen as confirmation of its identity as higher plant ferredoxin sulfite reductase.
Assuntos
Proteínas de Arabidopsis , Arabidopsis/enzimologia , Arabidopsis/genética , DNA Complementar/isolamento & purificação , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Plastídeos/enzimologia , Plastídeos/genética , Sequência de Aminoácidos , Sequência de Bases , Cianobactérias/enzimologia , Escherichia coli/enzimologia , Dados de Sequência Molecular , Peso Molecular , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Homologia de Sequência , Sulfito Redutase (Ferredoxina) , Sulfito Redutase (NADPH)RESUMO
A cDNA clone with an open reading frame of 831 nucleotides was isolated from a lambda ZapII-library of Arabidopsis thaliana. The nucleotide sequence of the cDNA is homologous to the APS-kinase genes from enterobacteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53.2%), Rhizobium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14:57.1%). The polypeptide deduced from the plant APS-kinase cDNA is comprised of 276 amino acid residues with a molecular weight of 29,790. It contains an N-terminal extension of 77 amino acids. This extension includes a putative transit peptide of 37 residues separated from the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26,050 is predicted for the processed protein. The relatedness between bacterial, fungal and plant APS-kinase polypeptides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. meliloti), and 50.3% (Azospirillum brasilense). The plant polypeptide contains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxxGK. Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif described for PAPS dependent enzymes. The identity of the gene was confirmed by analyzing the function of the gene product. The putative transit peptide was deleted by PCR and the truncated gene was expressed in a pTac1 vector system. A polypeptide of MW 25761 could be induced by IPTG. The gene product was enzymatically active as APS-kinase. It produced PAPS from APS and ATP--the absence of ATP but supplemented with thiols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransferase is not a separate enzyme but identical with APS-kinase.