RESUMEN
Non-Hodgkin's lymphoma is a common type of cancer, whose most common site of extranodal involvement is the gastrointestinal tract. However, primary presentation in the pancreas remains uncommon. Among cases with pancreatic involvement, the disease is often found in the head and rarely in the tail. Here, we present a case of a 56-year-old male patient with acute epigastric pain, early satiety, and abdominal distention. CT imaging showed a mass of the pancreatic tail with surrounding lymphadenopathy, concerning lymphoma. Endoscopic ultrasound-guided fine needle aspiration (EUS-guided FNA) diagnosed mature B-cell lymphoma, meeting novel diagnostic criteria for the rare diagnosis of primary pancreatic lymphoma (PPL).
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BACKGROUND: Damage to the adult central nervous system often leads to long-term disruptions in function due to the limited capacity for neurological recovery. The central nervous system of the Mediterranean field cricket, Gryllus bimaculatus, shows an unusual capacity for compensatory plasticity, most obviously in the auditory system and the cercal escape system. In both systems, unilateral sensory disruption leads the central circuitry to compensate by forming and/or strengthening connections with the contralateral sensory organ. While this compensatory plasticity in the auditory system relies on robust dendritic sprouting and novel synapse formation, the compensatory plasticity in the cercal escape circuitry shows little obvious dendritic sprouting and instead may rely on shifts in excitatory and inhibitory synaptic strength. RESULTS: In order to better understand what types of molecular pathways might underlie this compensatory shift in the cercal system, we used a multiple k-mer approach to assemble a terminal ganglion transcriptome that included ganglia collected one, three, and 7 days after unilateral cercal ablation in adult, male animals. We performed differential expression analysis using EdgeR and DESeq2 and examined Gene Ontologies to identify candidates potentially involved in this plasticity. Enriched GO terms included those related to the ubiquitin-proteosome protein degradation system, chromatin-mediated transcriptional pathways, and the GTPase-related signaling system. CONCLUSION: Further exploration of these GO terms will provide a clearer picture of the processes involved in compensatory recovery of the cercal escape system in the cricket and can be compared and contrasted with the distinct pathways that have been identified upon deafferentation of the auditory system in this same animal.
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Gryllidae , Animales , Sistema Nervioso Central , Gryllidae/genética , Interneuronas , MasculinoRESUMEN
BACKGROUND: Where a neuron is positioned in the brain during development determines neuronal circuitry and information processing needed for normal brain function. When aberrations in this process occur, cognitive disorders may result. Patients diagnosed with schizophrenia have been reported to show altered neuronal connectivity and heterotopias. To elucidate pathways by which this process occurs and become aberrant, we have chosen to study the long isoform of nitric oxide synthase 1 adaptor protein (NOS1AP), a protein encoded by a susceptibility gene for schizophrenia. METHODS: To determine whether NOS1AP plays a role in cortical patterning, we knocked down or co-overexpressed NOS1AP and a green fluorescent protein or red fluorescent protein (TagRFP) reporter in neuronal progenitor cells of the embryonic rat neocortex using in utero electroporation. We analyzed sections of cortex (ventricular zone, intermediate zone, and cortical plate [CP]) containing green fluorescent protein or red fluorescent protein TagRFP positive cells and counted the percentage of positive cells that migrated to each region from at least three rats for each condition. RESULTS: NOS1AP overexpression disrupts neuronal migration, resulting in increased cells in intermediate zone and less cells in CP, and decreases dendritogenesis. Knockdown results in increased migration, with more cells reaching the CP. The phosphotyrosine binding region, but not the PDZ-binding motif, is necessary for NOS1AP function. Amino acids 181 to 307, which are sufficient for NOS1AP-mediated decreases in dendrite number, have no effect on migration. CONCLUSIONS: Our studies show for the first time a critical role for the schizophrenia-associated gene NOS1AP in cortical patterning, which may contribute to underlying pathophysiology seen in schizophrenia.
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Proteínas Adaptadoras Transductoras de Señales/deficiencia , Movimiento Celular/genética , Neocórtex/citología , Células-Madre Neurales/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Animales Recién Nacidos , Células COS , Chlorocebus aethiops , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Mutación/genética , Células-Madre Neurales/metabolismo , Dominios PDZ/genética , Embarazo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Transgénicas , Transfección , Proteína Fluorescente RojaRESUMEN
The Co centre in the title compound, [Co(C(14)H(11)N(2)O(2))(2)Cl(C(3)H(4)N(2))]·0.5H(2)O, shows a slightly distorted octa-hedral coordination geometry. The glyoximate units of the mol-ecule are linked by O-Hâ¯O hydrogen bonds with the H atom almost in the middle of the two O atoms. The crystal packing is stabilized through inter-molecular N-Hâ¯O, N-Hâ¯N and O-Hâ¯Cl hydrogen bonds. The uncoordinated water mol-ecule shows half-occupation.
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The genetic basis of bipolar disorder (BPD) and schizophrenia (SCZ) has been established through numerous clinical and molecular studies. Although often considered separate nosological entities, evidence now suggests that the two syndromes may share some genetic liability. Recent studies have used a composite phenotype (psychosis) that includes BPD, SCZ, psychosis not otherwise specified, and schizoaffective disorder, to identify shared susceptibility loci. Several chromosomal regions are reported to be shared between these syndromes (18p, 6q, 10p, 13q, 22q). As a part of our endeavor to scan these regions, we report a positive linkage and association finding at 18p11.2 for psychosis. Two-point linkage analysis performed on a series of 52 multiplex pedigrees with 23 polymorphic markers yielded a LOD score of 2.02 at D18S37. An independent set of 159 parent offspring trios was used to confirm this suggestive finding. The TDT analysis yielded support for association between the marker D18S453 and the disease allele (chi2 = 4.829, P < 0.028). This region has been implicated by several studies on BPD [Sjoholt et al. (2004); Mol Psychiatry 9(6):621-629; Washizuka et al. (2004); Biol Psychiatry 56(7):483-489; Pickard et al. (2005); Psychiatr Genet 15(1):37-44], SCZ [Kikuchi et al. (2003); J Med Dent Sci 50(3):225-229; Babovic-Vuksanovic et al. (2004); Am J Med Genet 124(3):318-322] and also as a shared region between the two diseases [Ishiguro et al. (2001); J Neural Transm 108(7):849-854; Reyes et al. (2002); Mol Psychiatry 7(4):337-339; Craddock et al. (2005); J Med Genet 42(3):193-204]. Our findings provide an independent validation of the above reports, and suggest the presence of susceptibility loci for psychoses in this region.
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Cromosomas Humanos Par 18/genética , Predisposición Genética a la Enfermedad , Desequilibrio de Ligamiento/genética , Polimorfismo Genético , Trastornos Psicóticos/genética , Esquizofrenia/genética , Genotipo , Humanos , India , Escala de Lod , LinajeRESUMEN
The voltage- and Ca(2+)-activated K(+) (K(V,Ca)) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K(V,Ca) alpha-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K(V,Ca) beta-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.
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Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Animales , Calcio/fisiología , Línea Celular , Membrana Celular/fisiología , Centrifugación por Gradiente de Densidad , Perros , Glicosilación , Humanos , Riñón , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Potenciales de la Membrana/fisiología , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Canales de Potasio/química , Canales de Potasio/genética , Subunidades de Proteína , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Large conductance voltage and Ca(2+)-activated K(+) (MaxiK) channels couple intracellular Ca(2+) with cellular excitability. They are composed of a pore-forming alpha subunit and modulatory beta subunits. The pore blockers charybdotoxin (CTx) and iberiotoxin (IbTx), at nanomolar concentrations, have been invaluable in unraveling MaxiK channel physiological role in vertebrates. However in mammalian brain, CTx-insensitive MaxiK channels have been described [Reinhart, P. H., Chung, S. & Levitan, I. B. (1989) Neuron 2, 1031-1041], but their molecular basis is unknown. Here we report a human MaxiK channel beta-subunit (beta4), highly expressed in brain, which renders the MaxiK channel alpha-subunit resistant to nanomolar concentrations of CTx and IbTx. The resistance of MaxiK channel to toxin block, a phenotype conferred by the beta4 extracellular loop, results from a dramatic ( approximately 1,000 fold) slowdown of the toxin association. However once bound, the toxin block is apparently irreversible. Thus, unusually high toxin concentrations and long exposure times are necessary to determine the role of "CTx/IbTx-insensitive" MaxiK channels formed by alpha + beta4 subunits.
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Encéfalo/fisiología , Caribdotoxina/farmacología , Neuronas/fisiología , Péptidos/farmacología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Femenino , Glicosilación , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Canales de Potasio/química , Canales de Potasio/genética , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , ARN Mensajero/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Xenopus laevisRESUMEN
Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane beta subunit (beta2) that yields inactivating MaxiK currents on coexpression with the pore-forming alpha subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the beta2 subunit abolished inactivation of the alpha subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle beta1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the beta2 subunit causes inactivation of the MaxiK channel alpha subunit by occluding its K+-conducting pore resembling the inactivation caused by the "ball" peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different beta subunits.
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Calcio/fisiología , Neuronas/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/química , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Bases de Datos como Asunto , Etiquetas de Secuencia Expresada , Femenino , Glicosilación , Humanos , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Sustancias Macromoleculares , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Oocitos/fisiología , Especificidad de Órganos , Canales de Potasio/genética , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Voltage- and Ca2+-sensitive K+ (MaxiK) channels play key roles in controlling neuronal excitability and vascular tone. We cloned and analyzed human and rodent genes for the modulatory beta subunit, KCNMB1. The human and mouse beta-subunit genes are approximately 11 and approximately 9 kb in length, respectively, and have a four exon-three intron structure. Primer extension assay localized the transcription initiation site at 442 (human) or 440 (mouse) bp upstream of the translation initiation codon, agreeing with the transcript size in Northern blots. Both genes have a TATA-less putative promoter region, with a transcription initiator-like region, and motifs characteristic of regulated promoters, including muscle-specific enhancing factors-1 and -2. Consistent with a tissue-specific expression of KCNMB1, regulated at the transcriptional level, beta-subunit transcripts are abundant in smooth muscle and heart, but scarce in lymphatic tissues, brain, and liver. Expressed rat and mouse beta subunits increase the apparent Ca2+ sensitivity of the human MaxiK channel alpha subunit.
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Canales de Potasio Calcio-Activados , Canales de Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/química , Clonación Molecular , ADN , Regulación de la Expresión Génica , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Ratones , Datos de Secuencia Molecular , Canales de Potasio/química , Canales de Potasio/fisiología , Regiones Promotoras Genéticas , Conformación Proteica , Ratas , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
We investigated the role of individual charged residues of the S4 region of a MaxiK channel (hSlo) in channel gating. We measured macroscopic currents induced by wild type (WT) and point mutants of hSlo in inside-out membrane patches of Xenopus laevis oocytes. Of all the residues tested, only neutralizations of Arg-210 and Arg-213 were associated with a reduction in the number of gating charges as determined using the limiting slope method. Channel activation in WT and mutant channels was interpreted using an allosteric model. Mutations R207Q, R207E, and R210N facilitated channel opening in the absence of Ca2+; however, this facilitation was not observed in the channels Ca2+-bound state. Mutation R213Q behaved similarly to the WT channel in the absence of Ca2+, but Ca2+ was unable to stabilize the open state to the same extent as it does in the WT. Mutations R207Q, R207E, R210N, and R213Q reduced the coupling between Ca2+ binding and channel opening when compared with the WT. Mutations L204R, L204H, Q216R, E219Q, and E219K in the S4 domain showed a similar phenotype to the WT channel. We conclude that the S4 region in the hSlo channel is part of the voltage sensor and that only two charged amino acid residues in this region (Arg-210 and Arg-213) contribute to the gating valence of the channel.
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Calcio/metabolismo , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Activación del Canal Iónico , Datos de Secuencia Molecular , Mutación Puntual , Canales de Potasio/química , Canales de Potasio/genética , Xenopus laevisRESUMEN
Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels.
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GMP Cíclico/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/metabolismo , Proteínas Quinasas/metabolismo , Animales , Clonación Molecular , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Membrana Dobles de Lípidos , Fosforilación , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Proteínas Quinasas/genética , XenopusRESUMEN
Large-conductance, voltage-, and Ca(2+)-sensitive K(+) (maxi-K(Ca)) channels regulate neuronal and smooth muscle excitability. Their pore-forming alpha-subunit shows similarities with voltage-gated channels and indeed can open in the practical absence of Ca(2+). The NH(2) terminus is unique, with a seventh transmembrane segment involved in beta-subunit modulation. The long COOH terminus is implied in Ca(2+) modulation.
RESUMEN
1. Human large-conductance voltage- and calcium-sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore-forming subunit, alpha, and a modulatory subunit, beta. Expression of the beta subunit induces dramatic changes in alpha subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS-I) to upregulate the channel. 2. The functional coupling of maxi KCa channel alpha and beta subunits in freshly dissociated human coronary smooth muscle cells was assessed. To distinguish maxi KCa currents modulated by the beta subunit, we examined (a) their apparent Ca2+ sensitivity, as judged from the voltage necessary to half-activate the channel (V1/2), and (b) their activation by DHS-I. 3. In patches with unitary currents, the majority of channels were half-activated near -85 mV at 18 microM Ca2+, a value similar to that obtained when the human KCa channel alpha (HSLO) and beta (HKV,Ca beta) subunits are co-expressed. A small number of channels half-activated around 0 mV, suggesting the activity of the alpha subunit alone. 4. The properties of macroscopic currents were consistent with the view that most pore-forming alpha subunits were coupled to beta subunits, since the majority of currents had values for V1/2 near to -90 mV, and currents were potentiated by DHS-I. 5. We conclude that in human coronary artery smooth muscle cells, most maxi KCa channels are composed of alpha and beta subunits. The higher Ca2+ sensitivity of maxi KCa channels, resulting from their coupling to beta subunits, suggests an important role of this channel in regulating coronary tone. Their massive activation by micromolar Ca2+ concentrations may lead to a large hyperpolarization causing profound changes in coronary blood flow and cardiac function.
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Vasos Coronarios/química , Músculo Liso Vascular/química , Ácido Oleanólico/análogos & derivados , Canales de Potasio Calcio-Activados , Canales de Potasio/química , Canales de Potasio/fisiología , Adolescente , Adulto , Anciano , Animales , Antineoplásicos Fitogénicos/farmacología , Calcio/fisiología , Niño , Vasos Coronarios/fisiología , Estimulación Eléctrica , Femenino , Humanos , Activación del Canal Iónico/fisiología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oocitos/química , Oocitos/fisiología , Técnicas de Placa-Clamp , Saponinas/farmacología , Triterpenos/farmacología , Xenopus laevisRESUMEN
Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers beta subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and "in vivo" reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.
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Canales de Potasio Calcio-Activados , Canales de Potasio/química , Secuencia de Aminoácidos , Animales , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Citosol/química , Citosol/metabolismo , Femenino , Humanos , Técnicas In Vitro , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Modelos Moleculares , Datos de Secuencia Molecular , Oocitos/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Biosíntesis de Proteínas , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The pore-forming alpha subunit of large conductance voltage- and Ca(2+)-sensitive K (MaxiK) channels is regulated by a beta subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming alpha subunit necessary for beta-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to beta-subunit modulation, and analyzed the topology of the alpha subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel alpha subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers beta-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.
Asunto(s)
Membrana Celular/fisiología , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Drosophila , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Proteínas Recombinantes de Fusión/fisiología , Análisis de SecuenciaRESUMEN
KV,Ca beta subunit dramatically increases the apparent calcium sensitivity of the alpha subunit of MaxiK channels when probed in the micromolar [Ca2+]i range. Analysis in a wide range of [Ca2+]i revealed that this functional coupling is exquisitely modulated by [Ca2+]i. Ca2+ ions switch MaxiK alpha+beta complex into a functionally coupled state at concentrations beyond resting [Ca2+]i. At [Ca2+] < or = 100 nM, MaxiK activity becomes independent of Ca2+, is purely voltage-activated, and its functional coupling with its beta subunit is released. The functional switch develops at [Ca2+]i that occur during cellular excitation, providing the molecular basis of how MaxiK channels regulate smooth muscle excitability and neurotransmitter release.
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Calcio/fisiología , Activación del Canal Iónico/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Cationes Bivalentes , Bovinos , Femenino , Humanos , Activación del Canal Iónico/efectos de los fármacos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Datos de Secuencia Molecular , Miometrio , Oocitos , Canales de Potasio/genética , Homología de Secuencia de Aminoácido , Xenopus laevisRESUMEN
Developmental changes in cellular Ca(2+)-transport mechanisms were studied in chick heart by determining cellular Ca(2+)-uptake and Na+,Ca(2+)-exchange activity in freshly isolated ventricular tissues of embryonic (5-18 days old), newborn (1-2 days old) and young adult (90-100 days old) heart by monitoring 45Ca influx. Ca(2+)-ATPase activity was determined in microsomal fractions at different stages of development. The Ca(2+)-uptake (per g wet tissue weight) increased with the development of embryonic as well as post-hatch chick heart, reaching a maximum in the young adult chicken. The overall increase in Ca(2+)-uptake, from embryonic day 5 to young-adult stage, was more than 3 fold. The Na+,Ca(2+)-exchange activity, determined as Na(+)-gradient-induced Ca(2+)-uptake in presence of either ouabain or zero [Na+]0, showed a 6-fold increase during development of heart from the embryonic day 5 to the young adult stage. Amiloride, an inhibitor of Na+,Ca(2+)-exchange, caused a dose-dependent reduction in a ouabain-induced rise in 45Ca influx at different stages of development. The inhibitory effect of amiloride was, however, greater during later stages of development. A progressive increase in Ca(2+)-ATPase activity was also seen during development. Ca(2+)-ATPase exhibited about a 4-fold increase in activity from embryonic day 7 to the young adult. The concomitant increase in Ca(2+)-uptake, Na+,Ca(2+)-exchange and Ca(2+)-ATPase activities suggests age-dependent changes in Ca(2+)-transport and storage systems of developing heart during embryogenesis and post-embryonic life. During embryogenesis the developmental increase in Na+,Ca(2+)-exchange activity was greater than that during post-hatch development of heart. However, the increase in Ca(2+)-ATPase activity was greater during post-hatch development than during embryogenesis. It is suggested that Na+,Ca(2+)-exchange and Ca(2+)-ATPase play a prominent role in maintaining cellular Ca2+ homeostasis during embryogenesis and after hatching.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Miocardio/metabolismo , Sodio , Animales , Animales Recién Nacidos , Embrión de Pollo , Pollos , Corazón/embriología , Corazón/crecimiento & desarrollo , Técnicas In Vitro , Miocardio/enzimología , Intercambiador de Sodio-CalcioRESUMEN
The effects of four major types of organic Ca2+ channel blockers, verapamil, nifedipine, diltiazem and fendiline and of tetrodotoxin (TXX), a fast Na+ channel blocker, on the action potential (AP) of freshly isolated 3-day-old embryonic chick ventricle (3d ECV) were investigated to resolve the controversy about the ionic basis of upstroke. The APs were characterized by a maximum diastolic potential (MDP) of -60 mV, an overshoot (Eov) of 16 mV and a maximum upstroke velocity (+Vmax) of 42 V s-1. All four Ca2+ channel blockers (0.1-40 microM) and TTX (0.1-80 nM) produced a dose-dependent reduction in +Vmax and Eov. MDP was also reduced by Ca2+ channel blockers in a dose-dependent manner but was unaffected by TTX. A significant linear correlation between MDP and +Vmax was observed for verapamil (r = 0.99), nifedipine (r = 0.99), diltiazem (r = 0.96) and fendiline (r = 0.98). Surprisingly, all Ca2+ channel blockers produced a dose-dependent positive chronotropic effect leading to cessation of firing at high doses (20-40 microM). In preparations becoming quiescent with high doses of verapamil (20-40 microM), elevated extracellular concentrations of Ca2+ (up to 9.6 nM) and isoproterenol (0.5-40 microM) failed to restore spontaneous APs. Electrical stimulation also failed to elicit APs in preparations inhibited by verapamil, diltiazem and fendiline. The inhibition of +Vmax by TTX demonstrates that fast Na+ channels were involved in the upstroke of AP in 3d ECV. Voltage-dependent inactivation of fast Na+ channels during depolarization (reduction in MDP) by the Ca2+ channel blockers explains their inhibitory effect on +Vmax and indicates that L-type Ca2+ channels had no significant role in the upstroke. A positive chronotropic effect of the Ca2+ channel blockers further suggests that slow Ca2+ channels are not involved in automaticity in freshly isolated 3d ECV.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Función Ventricular/fisiología , Potenciales de Acción , Animales , Calcio/farmacología , Embrión de Pollo , Diltiazem/farmacología , Relación Dosis-Respuesta a Droga , Fendilina/farmacología , Ventrículos Cardíacos/embriología , Técnicas In Vitro , Isoproterenol/farmacología , Nifedipino/farmacología , Análisis de Regresión , Tetrodotoxina/farmacología , Función Ventricular/efectos de los fármacos , Verapamilo/farmacologíaRESUMEN
Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism.