RESUMEN
Assembly of transmembrane (TM) domains is a critical step in the function of membrane proteins, and therefore, determining the amino acid motifs that mediate this process is important. Studies along this line have shown that the GXXXG motif is involved in TM assembly. In this study we characterized the minimal dimerization motif in the bacterial Tar-1 homodimer TM domain, which does not contain a GXXXG sequence. We found that a short polar motif QXXS is sufficient to induce stable TM-TM interactions. Statistical analysis revealed that this motif appears to be significantly over-represented in a bacterial TM data base compared with its theoretical expectancy, suggesting a general role for this motif in TM assembly. A truncated short TM peptide (9 residues) that contains the QXXS motif interacted slightly with the wild-type Tar-1. However, the same short TM peptide regained wild-type-like activity when conjugated to an octanoyl aliphatic moiety. Biophysical studies indicated that this modification compensated for the missing hydrophobicity, stabilized alpha-helical structure, and enabled insertion of the peptide into the membrane core. These findings serve as direct evidence that even a short peptide containing a minimal recognition motif is sufficient to inhibit the proper assembly of TM domains. Interestingly, electron microscopy revealed that above the critical micellar concentration, the TM lipopeptide forms a network of nanofibers, which can serve for the slow release of the active lipopeptide.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Dimerización , Escherichia coli , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas , Proteínas de la Membrana/química , Fragmentos de Péptidos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Aminoácidos/biosíntesis , Receptores de Aminoácidos/químicaRESUMEN
Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis. This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system. A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals. While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear. This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6'. To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6. The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example). Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand. The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation. Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound. Analogous CPU elements may be found in other receptors and signaling proteins.
Asunto(s)
Ácido Aspártico/metabolismo , Proteínas de Escherichia coli , Proteínas Quinasas/metabolismo , Receptores de Aminoácidos/química , Receptores de Aminoácidos/fisiología , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/fisiología , Sitios de Unión , Quimiotaxis/genética , Escherichia coli , Proteínas de la Membrana/fisiología , Proteínas Quimiotácticas Aceptoras de Metilo , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Estructura Secundaria de Proteína , Receptores de Aminoácidos/biosíntesis , Receptores de Aminoácidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/síntesis química , Salmonella typhimuriumRESUMEN
It has been postulated that, in the adult visual cortex, visual inputs modulate levels of mRNAs coding for neurotransmitter receptors in an activity-dependent manner. To investigate this possibility, we performed a monocular enucleation in adult rabbits and, 15 days later, collected their left and right visual cortices. Levels of mRNAs coding for voltage-activated sodium channels, and for receptors for kainate/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA), and glycine were semiquantitatively estimated in the visual cortices ipsilateral and contralateral to the lesion by the Xenopus oocyte/voltage-clamp expression system. This technique also allowed us to study some of the pharmacological and physiological properties of the channels and receptors expressed in the oocytes. In cells injected with mRNA from left or right cortices of monocularly enucleated and control animals, the amplitudes of currents elicited by kainate or AMPA, which reflect the abundance of mRNAs coding for kainate and AMPA receptors, were similar. There was no difference in the sensitivity to kainate and in the voltage dependence of the kainate response. Responses mediated by NMDA, GABA, and glycine were unaffected by monocular enucleation. Sodium channel peak currents, activation, steady-state inactivation, and sensitivity to tetrodotoxin also remained unchanged after the enucleation. Our data show that mRNAs for major neurotransmitter receptors and ion channels in the adult rabbit visual cortex are not obviously modified by monocular deafferentiation. Thus, our results do not support the idea of a widespread dynamic modulation of mRNAs coding for receptors and ion channels by visual activity in the rabbit visual system.
Asunto(s)
Activación del Canal Iónico , Receptores de Aminoácidos/biosíntesis , Canales de Sodio/biosíntesis , Visión Monocular/fisiología , Corteza Visual/metabolismo , Vías Aferentes/cirugía , Animales , Relación Dosis-Respuesta a Droga , Electrofisiología/métodos , Enucleación del Ojo , Masculino , Oocitos , ARN Mensajero/aislamiento & purificación , Conejos , Receptores de Aminoácidos/genética , Bloqueadores de los Canales de Sodio , Canales de Sodio/genética , Tetrodotoxina/farmacología , XenopusRESUMEN
We addressed the question of whether glial cells in intact white matter tracts express neurotransmitter receptors and we used Ca+2 signalling as a probe to detect the receptor activation. Corpus callosum slices from postnatal mice were bulk-loaded with the Ca+2-sensitive fluorescent dye fluo-3, and confocal microscopy was used to measure Ca+2 transients in response to neuroligands. Glial cell bodies were intensely dye-loaded and could be discriminated from the diffuse fluorescence of axons. Subpopulations of glial cells from slices obtained at postnatal days 3 to 7 responded with Ca+2 signals to ATP, glutamate, histamine, GABA, norepinephrine, serotonin, angiotensin II, bradykinin, and substance P. These subpopulations showed a distinct overlap; cells which were responsive to substance P always showed Ca+2 signalling in response to histamine, ATP, GABA and high K+ (membrane depolarization). GABA-responsive cells almost always showed a [Ca+2]i increase after membrane depolarization. In brain slices from postnatal day 11 to 18 animals, the Ca+2 responses were evident for glutamate, ATP, and norepinephrine, while GABA, substance P, serotonin, histamine, or angiotensin II rarely elicited a response. This study demonstrates that white matter glial cells in slices exhibit a large repertoire of neurotransmitter responses linked to Ca+2 signalling and that these receptor systems are differentially distributed on sub-populations of glial cells.
Asunto(s)
Calcio/fisiología , Cuerpo Calloso/fisiología , Neuroglía/fisiología , Transducción de Señal/fisiología , Animales , Astrocitos/metabolismo , Linaje de la Célula , Cuerpo Calloso/citología , Electrofisiología , Fluorometría , Potenciales de la Membrana/fisiología , Ratones , Oligodendroglía/metabolismo , Técnicas de Placa-Clamp , Receptores de Aminoácidos/biosíntesis , Receptores de Neuropéptido/biosíntesis , Receptores de Neurotransmisores/biosíntesis , Receptores Purinérgicos P1/biosíntesis , Receptores Purinérgicos P2/biosíntesisRESUMEN
To determine the role in transmembrane signaling of the N-terminal peptide of the first transmembrane region of the aspartate receptor, it was subjected to extensive mutagenesis. Drastic changes did not alter the chemotactic ability of the receptor to aspartate significantly. Thus the cytoplasmic N terminus of the first transmembrane region does not play an essential role in transmembrane signaling, and the entire signal that is transmitted to the cytoplasmic domain must be sent through the second transmembrane region. This eliminates the models requiring an interaction of this N-terminal peptide with the remaining cytoplasmic portion of the receptor.
Asunto(s)
Quimiotaxis , Escherichia coli/fisiología , Receptores de Aminoácidos/metabolismo , Ácido Aspártico/metabolismo , Membrana Celular/fisiología , Citoplasma/fisiología , Escherichia coli/genética , Cinética , Mutagénesis , Plásmidos , Receptores de Aminoácidos/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transducción de SeñalRESUMEN
Excitatory amino acids (EAA) acting on N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate receptors play an important role in synaptic transmission in the spinal cord. Quantitative autoradiography and physiological experiments suggest that NMDA receptors are localized mainly in lamina II while kainate and AMPA receptors are found on both dorsal and ventral horn neurons. However the cell types expressing EAA receptors and their laminar distribution is not known. We have used a cobalt uptake method to study the morphology and distribution of spinal cord neurons expressing AMPA, kainate, or NMDA excitatory amino acid receptors in the lumbar enlargement of the rat spinal cord. The technique involved superfusion of hemisected spinal cords of 14 day-old rat pups in vitro with excitatory amino acid receptor ligands in the presence of CoCl2. Cobalt has been shown to enter cells through ligand-gated ion channels in place of Ca2+. Cells which accumulated cobalt ions following activation by ionotropic excitatory amino acid receptors were visualized histochemically. The cobalt uptake generated receptor-specific labeling of cells, as the NMDA receptor antagonist D-(-)-2-amino-(5)-phosphonovaleric acid (D-AP-5) (20 microM) blocked the NMDA, but not kainate-induced cobalt uptake. The kainate-induced cobalt labeling was reduced by the non-selective excitatory amino acid receptor antagonist kynurenic acid (4 mM). Passive opening of the voltage-gated Ca(2+)-channels by KCl (50 mM) did not result in cobalt uptake, indicating that cobalt enters the cells through ligand-gated Ca(2+)-channels. AMPA (500 microM), kainate (500 microM), or NMDA (500 microM) each induced cobalt uptake with characteristic patterns and distributions of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal staining. Overall, kainate induced cobalt uptake in the greatest number of neuronal perikarya while NMDA-induced uptake was the lowest. AMPA and kainate, but not NMDA superfusion, resulted in cobalt labeling of glial cells. Our results show that the cobalt uptake technique is a useful way to study the morphology and distribution of cells expressing receptors with ligand-gated Ca2+ channels.
Asunto(s)
Cobalto/metabolismo , Neuronas/metabolismo , Receptores de Aminoácidos/biosíntesis , Médula Espinal/metabolismo , Animales , Canales de Calcio/metabolismo , Fura-2 , Activación del Canal Iónico/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/ultraestructura , Neuronas/ultraestructura , Ratas , Ratas Sprague-Dawley , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/biosíntesis , Receptores de Aminoácidos/antagonistas & inhibidores , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de Ácido Kaínico/biosíntesis , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/biosíntesis , Médula Espinal/citología , Médula Espinal/ultraestructuraRESUMEN
The 4 glutamate residues that are the sites of methylation in the aspartate receptor, which mediates bacterial chemotaxis, were systematically mutated to aspartate residues. None of the aspartate residues were methylated. In each case, the presence of the aspartate mutations altered the methylation rates at glutamate residues on the same receptor. Methylation was nearly eliminated at residues two turns of alpha helix, in the N-terminal direction, from a site of mutation, whereas less severe changes occurred at other positions. The methyltransferase apparently must contact a glutamate or glutamine residue two turns of helix in the C-terminal direction from the site of modification in order to methylate that position at a maximal rate. Mutations from glutamate to aspartate at any of the four sites also appear to alter the methylation rate at the other sites through a change in the structure of the receptor. The aspartate mutations did not substantially alter the affinity of the methyltransferase for the receptor.
Asunto(s)
Escherichia coli/metabolismo , Metiltransferasas/metabolismo , Receptores de Aminoácidos/metabolismo , Secuencia de Aminoácidos , Ácido Aspártico/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Escherichia coli/enzimología , Genes Bacterianos , Cinética , Metilación , Metiltransferasas/biosíntesis , Metiltransferasas/química , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Plásmidos , Receptores de Aminoácidos/biosíntesis , Receptores de Aminoácidos/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
A cDNA, HGl, encoding an inhibitory amino-acid receptor subunit has been cloned from a mixed egg population of the parasitic nematode Haemonchus contortus. The predicted amino-acid sequence of the subunit shows 24% to 32% homology with other vertebrate and invertebrate GABAA and glycine receptor subunits and has all the expected motifs for a member of the ligand-gated ion channel superfamily. When expressed in Xenopus oocytes HGl gives a small response to 1 mM glycine, but not to 1 mM GABA, glutamate, taurine or L-alanine.
Asunto(s)
Haemonchus/metabolismo , Canales Iónicos/biosíntesis , Oocitos/fisiología , Receptores de Aminoácidos/biosíntesis , Alanina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , ADN Complementario/análisis , Femenino , Expresión Génica , Ácido Glutámico/farmacología , Glicina/farmacología , Invertebrados , Canales Iónicos/fisiología , Sustancias Macromoleculares , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Óvulo/metabolismo , Receptores de Aminoácidos/efectos de los fármacos , Receptores de Aminoácidos/fisiología , Homología de Secuencia de Aminoácido , Taurina/farmacología , Vertebrados , Xenopus , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
In order to facilitate biochemical studies of cell-surface receptors, a plasmid allowing the expression of the periplasmic domain of the aspartate receptor from Salmonella typhimurium as a soluble periplasmic protein has been constructed. This 18-kDa protein is exported to the periplasm, where it may be extracted by mild osmotic lysis. This isolated domain behaves as a normal, soluble protein and has been purified to homogeneity by standard techniques. The purified periplasmic domain binds aspartate with a kD similar to that of the full-length receptor, and the binding occurs with negative cooperativity, i.e. the binding of one molecule of aspartate induces a conformational change that interferes with the binding of the second aspartate. Unlike the full-length receptor, the periplasmic domain undergoes a protein concentration- and aspartate-dependent monomer-dimer equilibrium. At low protein concentrations and in the absence of aspartate, the protein is monomeric. At higher protein concentrations or in the presence of saturating aspartate, the protein is dimeric. Two charge variants of the protein have been identified on native polyacrylamide gels. The more acidic form is blocked to Edman degradation, indicating that modification of the amino terminus of this protein can occur after cleavage of the signal peptide in the periplasm.
Asunto(s)
Ácido Aspártico/metabolismo , Receptores de Aminoácidos/aislamiento & purificación , Receptores de Aminoácidos/metabolismo , Salmonella typhimurium/metabolismo , Cromatografía , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cromatografía Liquida , Clonación Molecular , Durapatita , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Hidroxiapatitas , Cinética , Peso Molecular , Mutagénesis Insercional , Receptores de Aminoácidos/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Levels of mRNAs encoding neurotransmitter receptors in the visual cortex, lateral geniculate nucleus, and superior colliculus of the rabbit and rat, and properties of the receptors expressed, were studied using Xenopus laevis oocytes. mRNA extracted from these areas was injected into the oocytes, which then acquired functional receptors. Electrical recordings of neurotransmitter-induced membrane currents reflect the relative amounts of mRNAs encoding the corresponding receptors. Receptors to gamma aminobutyric acid (GABA), kainate, glutamate, and serotonin exhibited uniformly high levels of expression, whereas expression of receptors to glycine and N-methyl-D-aspartate was uniformly low. In contrast, the expression of receptors to acetylcholine and substance P was highly non-uniform. Expression of acetylcholine receptors was high in oocytes injected with mRNA from the visual cortex, low for the lateral geniculate nucleus, and very low or absent for the superior colliculus. Conversely, the currents elicited by substance P were large in oocytes injected with superior colliculus mRNA, but were small or absent in oocytes injected with mRNAs from the other regions. Immunohistochemical analysis, at the light and electron microscopic levels, was used to localize choline acetyltransferase, the acetylcholine-synthesizing enzyme, and substance P-containing synaptic boutons in the three visual areas. Their presence closely paralleled the potency of mRNAs coding for acetylcholine and substance P receptors. The ability of rat mRNA, from each visual area, to induce neurotransmitter receptors was similar to that observed in the corresponding rabbit mRNAs. In addition to the marked differential distribution of mRNA encoding neurotransmitter receptors in the visual system, our findings reveal the probable existence of as yet uncharacterized receptors, whose new molecular forms may be revealed by further study. Our results also provide the basic information required for subsequent studies on the effect of monocular deprivation on the expression of neurotransmitter receptors in the visual system.