RESUMEN
Polyamines (PAs) including putrescine (PUT), spermidine (SPD) and spermine (SPM) are small, versatile molecules with two or more positively charged amino groups. Despite their importance for almost all forms of life, their specific roles in molecular and cellular biology remain partly unknown. The molecular structures of PAs suggest two presumable biological functions: (i) as potential buffer systems and (ii) as interactants with poly-negatively charged molecules like nucleic acids. The present report focuses on the question, whether the molecular structures of PAs are essential for such functions, or whether other simple molecules like small peptides with closely spaced positively charged side chains might be suitable as well. Consequently, we created titration curves for PUT, SPD, and SPM, as well as for oligolysines like tri-, tetra-, and penta-lysine. None of the molecules provided substantial buffering capacity at physiological intracellular pH values. Apparently, the most important mechanism for intracellular pH homeostasis in neurons is not a buffer system but is provided by the actions of the sodium-hydrogen and the bicarbonate-chloride antiporters. In a similar approach we investigated the interaction with DNA by following the extinction at 260 nm when titrating DNA with the above molecules. Again, PUT and tri-lysine were not able to interact with herring sperm DNA, while SPD and SPM were. Obviously, the presence of several positively charged groups on its own is not sufficient for the interaction with nucleic acids. Instead, the precise spacing of these groups is necessary for biological activity.
Asunto(s)
ADN , Péptidos , Poliaminas , ARN , Concentración de Iones de Hidrógeno , ADN/química , ADN/metabolismo , Tampones (Química) , ARN/química , ARN/metabolismo , Poliaminas/química , Poliaminas/metabolismo , Péptidos/química , Péptidos/metabolismo , Animales , Espermidina/química , Espermidina/metabolismo , Espermina/química , Espermina/metabolismo , Putrescina/química , Putrescina/metabolismoRESUMEN
With a single gene encoding HV1 channel, proton channel diversity is particularly low in mammals compared to other members of the superfamily of voltage-gated ion channels. Nonetheless, mammalian HV1 channels are expressed in many different tissues and cell types where they exert various functions. In the first part of this review, we regard novel aspects of the functional expression of HV1 channels in mammals by differentially comparing their involvement in (1) close conjunction with the NADPH oxidase complex responsible for the respiratory burst of phagocytes, and (2) in respiratory burst independent functions such as pH homeostasis or acid extrusion. In the second part, we dissect expression of HV channels within the eukaryotic tree of life, revealing the immense diversity of the channel in other phylae, such as mollusks or dinoflagellates, where several genes encoding HV channels can be found within a single species. In the last part, a comprehensive overview of the biophysical properties of a set of twenty different HV channels characterized electrophysiologically, from Mammalia to unicellular protists, is given.
Asunto(s)
Canales Iónicos , Protones , Animales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Membrana Celular/metabolismo , Estallido Respiratorio , Eucariontes/metabolismo , Mamíferos/metabolismoRESUMEN
Recently, three proton channels (HV ) have been identified and characterized in Aplysia californica (AcHV 1-3). Focusing on AcHV 1 and AcHV 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified HV homologous channels in other molluscs: channels homologous to AcHV 1 and to AcHV 2 were found in 90 species (56 full-length sequences) and in 33 species (18 full-length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, HV 4. This new family has high homology to AcHV 1 and AcHV 2 and was identified only in bivalvian molluscs (13 species, 12 full-length sequences). Typically, these channels possess an extracellular S1-S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan HV 1 channels (~ 13 amino acids) and the much larger loops of molluscan HV 2 channels (> 65 amino acids). The characteristic voltage-sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, HV 4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgHV 4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgHV 4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH- and voltage-regulated gating. This work is the first description of a HV 4 type channel, adding a new member to the recently expanded family of proton channels.
Asunto(s)
Canales Iónicos , Protones , Animales , Canales Iónicos/metabolismo , Activación del Canal Iónico/fisiología , Aminoácidos , Arginina , Moluscos/genética , Moluscos/metabolismoRESUMEN
Voltage-gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage-gated proton channels (HV ) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV 1, AcHV 2, and AcHV 3) are distributed throughout the whole animal. Patch-clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV 1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV 2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV 3, which differs from AcHV 1 and AcHV 2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species.
Asunto(s)
Activación del Canal Iónico , Protones , Animales , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Arginina , Citosol/metabolismo , Mamíferos/metabolismoRESUMEN
Polyamines (PAs) are small, versatile molecules with two or more nitrogen-containing positively charged groups and provide widespread biological functions. Most of these aspects are well known and covered by quite a number of excellent surveys. Here, the present review includes novel aspects and questions: (1) It summarizes the role of most natural and some important synthetic PAs. (2) It depicts PA uptake from nutrition and bacterial production in the intestinal system following loss of PAs via defecation. (3) It highlights the discrepancy between the high concentrations of PAs in the gut lumen and their low concentration in the blood plasma and cerebrospinal fluid, while concentrations in cellular cytoplasm are much higher. (4) The present review provides a novel and complete scheme for the biosynthesis of Pas, including glycine, glutamate, proline and others as PA precursors, and provides a hypothesis that the agmatine pathway may rescue putrescine production when ODC knockout seems to be lethal (solving the apparent contradiction in the literature). (5) It summarizes novel data on PA transport in brain glial cells explaining why these cells but not neurons preferentially accumulate PAs. (6) Finally, it provides a novel and complete scheme for PA interconversion, including hypusine, putreanine, and GABA (unique gliotransmitter) as end-products. Altogether, this review can serve as an updated contribution to understanding the PA mystery.
Asunto(s)
Poliaminas , Espermina , Sistema Nervioso Central/metabolismo , Neuroglía/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Espermina/metabolismoRESUMEN
Voltage-gated proton channels (HV 1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about HV 1 channels in insects. A characteristic aspartate (Asp) that functions as the proton selectivity filter (SF) and the RxWRxxR voltage-sensor motif are conserved structural elements in HV 1 channels. By analysing Transcriptome Shotgun Assembly (TSA) databases, we found 33 polyneopteran species meeting these structural requirements. Unexpectedly, an unusual natural variation Asp to glutamate (Glu) at SF was found in Phasmatodea and Mantophasmatodea. Additionally, we analysed the expression and function of HV 1 in the phasmatodean stick insect Extatosoma tiaratum (Et). EtHV 1 is strongly expressed in nervous tissue and shows pronounced inward proton conduction. This is the first study of a natural occurring Glu within the SF of a functional HV 1 and might be instrumental in uncovering the physiological function of HV 1 in insects.
Asunto(s)
Canales Iónicos , Protones , Animales , Insectos/metabolismo , Canales Iónicos/genéticaRESUMEN
The striatum is the main input structure of the basal ganglia. Distinct striatal subfields are involved in voluntary movement generation and cognitive and emotional tasks, but little is known about the morphological and molecular differences of striatal subregions. The ventrolateral subfield of the striatum (VLS) is the orofacial projection field of the sensorimotor cortex and is involved in the development of orofacial dyskinesias, involuntary chewing-like movements that often accompany long-term neuroleptic treatment. The biological basis for this particular vulnerability of the VLS is not known. Potassium channels are known to be strategically localized within the striatum. In search of possible molecular correlates of the specific vulnerability of the VLS, we analyzed the expression of voltage-gated potassium channels in rodent and primate brains using qPCR, in situ hybridization, and immunocytochemical single and double staining. Here we describe a novel, giant, non-cholinergic interneuron within the VLS. This neuron coexpresses the vesicular GABA transporter, the calcium-binding protein parvalbumin (PV), and the Kv3.3 potassium channel subunit. This novel neuron is much larger than PV neurons in other striatal regions, displays characteristic electrophysiological properties, and, most importantly, is restricted to the VLS. Consequently, the giant striatal Kv3.3-expressing PV neuron may link compromised Kv3 channel function and VLS-based orofacial dyskinesias.
Asunto(s)
Discinesias , Parvalbúminas , Animales , Cuerpo Estriado/metabolismo , Discinesias/metabolismo , Interneuronas/metabolismo , Parvalbúminas/metabolismo , Canales de Potasio/metabolismo , Canales de Potasio Shaw/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos InhibidoresRESUMEN
The bed bug Cimex lectularius is a globally distributed human ectoparasite with fascinating biology. It has recently acquired resistance against a broad range of insecticides, causing a worldwide increase in bed bug infestations. The recent annotation of the bed bug genome revealed a full complement of neuropeptide and neuropeptide receptor genes in this species. With regard to the biology of C. lectularius, neuropeptide signaling is especially interesting because it regulates feeding, diuresis, digestion, as well as reproduction and also provides potential new targets for chemical control. To identify which neuropeptides are translated from the genome-predicted genes, we performed a comprehensive peptidomic analysis of the central nervous system of the bed bug. We identified in total 144 different peptides from 29 precursors, of which at least 67 likely present bioactive mature neuropeptides. C. lectularius corazonin and myosuppressin are unique and deviate considerably from the canonical insect consensus sequences. Several identified neuropeptides likely act as hormones, as evidenced by the occurrence of respective mass signals and immunoreactivity in neurohemal structures. Our data provide the most comprehensive peptidome of a Heteropteran species so far and in comparison suggest that a hematophageous life style does not require qualitative adaptations of the insect peptidome.
Asunto(s)
Chinches/química , Sistema Nervioso Central/química , Neuropéptidos/análisis , Animales , Infestaciones Ectoparasitarias , Genoma , Hormonas , Proteínas de Insectos , ProteómicaRESUMEN
BACKGROUND: Neuropeptides are key players in information transfer and act as important regulators of development, growth, metabolism, and reproduction within multi-cellular animal organisms (Metazoa). These short protein-like substances show a high degree of structural variability and are recognized as the most diverse group of messenger molecules. We used transcriptome sequences from the 1KITE (1K Insect Transcriptome Evolution) project to search for neuropeptide coding sequences in 24 species from the non-pterygote hexapod lineages Protura (coneheads), Collembola (springtails), Diplura (two-pronged bristletails), Archaeognatha (jumping bristletails), and Zygentoma (silverfish and firebrats), which are often referred to as "basal" hexapods. Phylogenetically, Protura, Collembola, Diplura, and Archaeognatha are currently placed between Remipedia and Pterygota (winged insects); Zygentoma is the sistergroup of Pterygota. The Remipedia are assumed to be among the closest relatives of all hexapods and belong to the crustaceans. RESULTS: We identified neuropeptide precursor sequences within whole-body transcriptome data from these five hexapod groups and complemented this dataset with homologous sequences from three crustaceans (including Daphnia pulex), three myriapods, and the fruit fly Drosophila melanogaster. Our results indicate that the reported loss of several neuropeptide genes in a number of winged insects, particularly holometabolous insects, is a trend that has occurred within Pterygota. The neuropeptide precursor sequences of the non-pterygote hexapods show numerous amino acid substitutions, gene duplications, variants following alternative splicing, and numbers of paracopies. Nevertheless, most of these features fall within the range of variation known from pterygote insects. However, the capa/pyrokinin genes of non-pterygote hexapods provide an interesting example of rapid evolution, including duplication of a neuropeptide gene encoding different ligands. CONCLUSIONS: Our findings delineate a basic pattern of neuropeptide sequences that existed before lineage-specific developments occurred during the evolution of pterygote insects.
Asunto(s)
Evolución Molecular , Proteínas de Insectos/genética , Insectos/genética , Neuropéptidos/genética , Secuencia de Aminoácidos , Animales , Artrópodos/clasificación , Artrópodos/genética , Proteínas de Insectos/química , Insectos/clasificación , Insectos/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/química , Filogenia , TranscriptomaRESUMEN
UNLABELLED: The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. DATABASE: Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722.
Asunto(s)
Membrana Celular/metabolismo , Insectos/metabolismo , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Potenciales de la Membrana/fisiología , Protones , Secuencia de Aminoácidos , Animales , Electrofisiología , Humanos , Insectos/clasificación , Datos de Secuencia Molecular , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
In vertebrates the "anti-reward-system" mainly is represented by the habenula and its medial (MHb) and especially lateral (LHb) complexes. Considerable knowledge has accumulated concerning subnuclear structures and connectivities of MHb and LHb subnuclei. The present investigation aimed to obtain novel information, whether MHb or LHb or their subnuclei display field-characteristic gene products, which may shed light on biological functions of these areas. Unfortunately this was not the case. Microarray analysis of mRNAs in microdissected habenular and thalamic control areas yielded expression values of 17,745 RNAs representing protein-coding genes, to which annotated gene names could be assigned. High relative values of genes with known expression in MHb, LHb or thalamus in the corresponding areas indicated a high precision of the microdissection procedure. Note that the present report emphasizes differences between and not absolute expression values in the selected regions. The present investigation disclosed that the LHb genetically is much closer related to the thalamus as compared to the MHb. The results presented here focuse on gene transcripts related to major transmitter systems, catecholamines and neuropeptides. Quite surprisingly, our data indicate potentially inhibitory effects of acetylcholine and glutamate in the habenula. In addition, the absence of the K-Cl co-transporter 2 supports a largely excitatory role of GABAergic transmission especially in the MHb. Furthermore, several G-protein related receptors (Gpr83, Gpr139, Gpr149, Gpr151, Gpr158) and many neuropeptides related to feeding are differentially expressed in the habenular region, indicating that its involvement in the regulation of food consumption and energy expenditure may have been underestimated so far.
Asunto(s)
Metabolismo Energético , Conducta Alimentaria , Expresión Génica , Habénula/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Acetilcolina/metabolismo , Animales , Cannabinoides/metabolismo , Dopamina/metabolismo , Perfilación de la Expresión Génica , Ácido Glutámico/metabolismo , Habénula/citología , Habénula/fisiología , Masculino , Neuropéptidos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Serotonina/metabolismo , Tálamo/metabolismo , Análisis de Matrices TisularesRESUMEN
The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host-symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human-bed bug and symbiont-bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite.
Asunto(s)
Chinches/genética , Infestaciones Ectoparasitarias , Conducta Alimentaria , Transferencia de Gen Horizontal/genética , Interacciones Huésped-Parásitos/genética , Resistencia a los Insecticidas/genética , Insecticidas , Animales , Genoma , Humanos , Análisis de Secuencia de ADNRESUMEN
Among all K2P channels, TASK-3 shows the most widespread expression in rat brain, regulating neuronal excitability and transmitter release. Using a recently purified and characterized polyclonal monospecific antibody against TASK-3, the entire rat brain was immunocytochemically analyzed for expression of TASK-3 protein. Besides its well-known strong expression in motoneurons and monoaminergic and cholinergic neurons, TASK-3 expression was found in most neurons throughout the brain. However, it was not detected in certain neuronal populations, and neuropil staining was restricted to few areas. Also, it was absent in adult glial cells. In hypothalamic areas, TASK-3 was particularly strongly expressed in the supraoptic and suprachiasmatic nuclei, whereas other hypothalamic nuclei showed lower protein levels. Immunostaining of hippocampal CA1 and CA3 pyramidal neurons showed strongest expression, together with clear staining of CA3 mossy fibers and marked staining also in the dentate gyrus granule cells. In neocortical areas, most neurons expressed TASK-3 with a somatodendritic localization, most obvious in layer V pyramidal neurons. In the cerebellum, TASK-3 protein was found mainly in neurons and neuropil of the granular cell layer, whereas Purkinje cells were only faintly positive. Particularly weak expression was demonstrated in the forebrain. This report provides a comprehensive overview of TASK-3 protein expression in the rat brain.
Asunto(s)
Química Encefálica , Encéfalo/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Encéfalo/citología , Inmunohistoquímica , Masculino , Canales de Potasio de Dominio Poro en Tándem/análisis , Transporte de Proteínas , Ratas , Ratas WistarRESUMEN
Polyamines are important regulators of basal cellular functions but also subserve highly specific tasks in the mammalian brain. With this respect, polyamines and the synthesizing and degrading enzymes are clearly differentially distributed in neurons versus glial cells and also in different brain areas. The synthesis of the diamine putrescine may be driven via two different pathways. In the "classical" pathway urea and carbon dioxide are removed from arginine by arginase and ornithine decarboxylase. The alternative pathway, first removing carbon dioxide by arginine decarboxlyase and then urea by agmatinase, may serve the same purpose. Furthermore, the intermediate product of the alternative pathway, agmatine, is an endogenous ligand for imidazoline receptors and may serve as a neurotransmitter. In order to evaluate and compare the expression patterns of the two gate keeper enzymes arginase and arginine decarboxylase, we generated polyclonal, monospecific antibodies against arginase-1 and arginine decarboxylase. Using these tools, we immunocytochemically screened the rat brain and compared the expression patterns of both enzymes in several brain areas on the regional, cellular and subcellular level. In contrast to other enzymes of the polyamine pathway, arginine decarboxylase and arginase are both constitutively and widely expressed in rat brain neurons. In cerebral cortex and hippocampus, principal neurons and putative interneurons were clearly labeled for both enzymes. Labeling, however, was strikingly different in these neurons with respect to the subcellular localization of the enzymes. While with antibodies against arginine decarboxylase the immunosignal was distributed throughout the cytoplasm, arginase-like immunoreactivity was preferentially localized to Golgi stacks. Given the apparent congruence of arginase and arginine decarboxylase distribution with respect to certain cell populations, it seems likely that the synthesis of agmatine rather than putrescine may be the main purpose of the alternative pathway of polyamine synthesis, while the classical pathway supplies putrescine and spermidine/spermine in these neurons.
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Encéfalo/enzimología , Carboxiliasas/metabolismo , Citoplasma/enzimología , Aparato de Golgi/enzimología , Neuronas/enzimología , Poliaminas/metabolismo , Agmatina/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Regulación Enzimológica de la Expresión Génica , Putrescina/metabolismo , Ratas , Espermina/metabolismo , Distribución TisularRESUMEN
Most of what we know today about the molecular constituents of the insect circadian clock was discovered in the fruit fly Drosophila melanogaster. Various other holometabolous and some hemimetabolous insects have also been examined for the presence of circadian genes. In these insects, per, tim1, and cry2 are part of a core feedback loop system. The proteins inhibit their own expression, leading to circadian oscillations of mRNA and proteins. Although cockroaches are successfully employed circadian model organisms, their clock genes are mostly unknown. Thus, we cloned putative circadian genes in Rhyparobia maderae (synonym Leucophaea maderae), showing the presence of period (per), timeless 1 (tim1), and mammalian-type cryptochrome (cry2). The expression levels of per, tim1, and cry2 in R. maderae were examined in various tissues and photoperiods employing quantitative PCR. In brains and excised accessory medullae, expression levels of rmPer, rmTim1, and rmCry2 oscillated in a circadian manner with peaks in the first half of the night. Oscillations mostly continued in constant conditions. In Malpighian tubules, no significant oscillations were found. In animals raised in different photoperiods (LD 18:6, 12:12, 6:18), the peak levels of rmPer, rmTim1, and rmCry2 expression adjusted with respect to the beginning of the scotophase. The daily mean of expression levels was significantly lower in short-day versus long-day animals. We suggest that rmPer, rmTim1, and rmCry2 are part of the Madeira cockroach nuclear circadian clock, which can adjust to different photoperiods.
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Relojes Circadianos , Cucarachas/genética , Criptocromos/genética , Proteínas de Insectos/genética , Proteínas Circadianas Period/genética , Secuencia de Aminoácidos , Animales , Cucarachas/metabolismo , Criptocromos/metabolismo , Retroalimentación Fisiológica , Expresión Génica , Genes de Insecto , Proteínas de Insectos/metabolismo , Masculino , Datos de Secuencia Molecular , Proteínas Circadianas Period/metabolismo , Fotoperiodo , Análisis de Secuencia de ADNRESUMEN
Motor neurons are large cholinergic neurons located in the brain stem and spinal cord. In recent years, a functional role for TASK channels in cellular excitability and vulnerability to anesthetics of motor neurons has been described. Using a polyclonal monospecific antibody against the tandem pore domain K(+) channel (K2P channel) TWIK-related acid-sensitive K(+) channel (TASK-3), we analyzed the expression of the TASK-3 protein in motor systems of the rat CNS. Immunocytochemical staining showed strong TASK-3 expression in motor neurons of the facial, trigeminal, ambiguus, and hypoglossal nuclei. Oculomotor nuclei (including trochlear and abducens nucleus) were also strongly positive for TASK-3. The parasympathetic Edinger-Westphal nucleus and dorsal vagal nucleus showed significant, but weaker expression compared with somato- and branchiomotoric neurons. In addition, motor neurons in the anterior horn of the spinal cord were also strongly labeled for TASK-3 immunoreactivity. Based on morphological criteria, TASK-3 was found in the somatodendritic compartment of motor neurons. Cellular staining using methyl green and immunofluorescence double-labeling with anti-vesicular acetylcholine transporter (anti-vAChT) indicated ubiquitous TASK-3 expression in motor neurons, whereas in other brain regions TASK-3 showed a widespread but not ubiquitous expression. In situ hybridization using a TASK-3 specific riboprobe verified the expression of TASK-3 in motor neurons at the mRNA level.
Asunto(s)
Neuronas Motoras/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Inmunohistoquímica , Masculino , Neuronas Motoras/citología , Transporte de Proteínas , Ratas , Ratas Wistar , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
The promising use of mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study of their physiology. Proliferation and differentiation of multipotent cells often involve changes in their metabolic state. In the present study, we analyzed the expression of ATP-sensitive potassium (K(ATP)) channels in MSC and upon in vitro differentiation. K(ATP) channels are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. Kir6.1, Kir6.2 and SUR2A were expressed in undifferentiated MSC, whereas SUR2B and SUR1 were not detected on cDNA and protein level. Upon adipogenic differentiation Kir6.1 and SUR2A showed a significant reduction of the amount of mRNA by 84% and 95%, respectively, whereas Kir6.2 expression was unchanged. Osteogenic differentiation strongly up-regulated Kir6.2 mRNA (28-fold) whereas Kir6.1 and SUR2A showed no significant change in expression. Quantitative Western blot analysis and immunofluorescence staining confirmed the elevated expression of Kir6.2 upon osteogenic differentiation. Taken together, expression changes of K(ATP) channels may contribute to in vitro differentiation of MSC and represent changes in the metabolic state of the developing tissue.
Asunto(s)
Diferenciación Celular , Células Madre Mesenquimatosas/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Adipocitos/citología , Adulto , Anciano , Western Blotting , Calcificación Fisiológica , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Humanos , Canales KATP/metabolismo , Potenciales de la Membrana , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Mensajero/metabolismo , Receptores de Droga/metabolismo , Fracciones Subcelulares , Receptores de Sulfonilureas , Regulación hacia Arriba , Adulto JovenRESUMEN
Large-conductance Ca(2+) -activated (BK) potassium channels are centrally involved in neurovascular coupling, immunity, and neural transmission. The ability to be synergistically activated by membrane depolarization, different ligands and intracellular Ca(2+) links intracellular signaling and membrane excitability. The diverse physiological functions of BK channels crucially depend on regulatory ß subunits. Although first studies characterized the neuronal distribution of BKß subunits in the rodent brain, it is largely unknown which ß subunit proteins are expressed in astrocytes and thus mediate these regulatory effects. We therefore analyzed the expression of BKß subunits in rat and mouse brain and glial cell cultures. A monospecific polyclonal antibody against the BKß4 channel subunit was raised, affinity-purified and extensively characterized. BKß4 and to a lesser degree BKß1 transcripts and protein were detected in several astrocytic populations and cultured cells. Particularly strong BKß4 immunostaining was detected in astrocytic progenitors derived from the subventricular zone. The overlapping expression of BKα and BKß4 in astrocytes implies a functional relationship and suggests that BKß4 is an important accessory ß subunit for astrocytic BK channels. In addition, BKß4 might exert effects independent of the α subunit as functional heterologous co-expression of Nav1.6 and BKß4 resulted in reduced Nav1.6 sodium currents. Thus, BKß4 expression in astrocytes likely participates in regulating astrocytic voltage gradients and maintaining K(+) homeostasis, hence enabling astrocytes to fulfill their complex regulatory influence on proper brain function.
Asunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Animales , Astrocitos/citología , Encéfalo/citología , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Células HEK293 , Homeostasis/genética , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Ratones , Ratones Endogámicos C57BL , Oocitos , Ratas , Ratas Wistar , Xenopus laevisRESUMEN
In rat brain slices, the Kv channel blocker 4-aminopyridine (4-AP) induces seizure-like events. This effect is absent in slices from chronic epileptic rats generated using the kainic acid model. The reason for this phenomenon remained elusive as an altered expression level of Kv channels was ruled out as a mechanism. We recently described that the Ile400Val RNA editing of Kv1.1 generates 4-AP-insensitive Kv1 channels (Kv1.1(I400V)). We therefore hypothesized that altered RNA editing levels account for the reduced ictogenic potency of 4-AP in chronic epileptic rats. We found fourfold increased RNA editing ratios in the entorhinal cortex of chronic epileptic animals compared to healthy control animals. Electrophysiologic recordings in Xenopus oocytes revealed that the observed increased Kv1.1(I400V) editing level can in fact lead to significant loss of 4-AP sensitivity. Our data suggest that altered Kv1.1(I400V) RNA editing contributes to the reduced ictogenic potential of 4-AP in chronic epileptic rats.