Two γ-aminobutyric acidA (GABAA) receptor chimeras were designed in order to elucidate the structural requirements for GABAA receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [3H]muscimol and [3H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA. 相似文献
Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication. 相似文献
Acetylcholine receptors were assayed with α-bugarotoxin on embryonic chick skeletal muscle growing in primary cell culture. Toxin was bound specifically to muscle cells and could be competed with D-tubocurarine. Two dissociation constants were obtained by equilibrium binding: 7.2 × 10?9M and 2.7 × 10?7M at 25°C. Two sets of rate constants were also obtained from dissociation kinetics. There are five times more low affinity sites on cells than high affinity sites. The average density of high-affinity receptors is about 200/μm2. A time course of toxin binding to receptors at 37°C vs 25°C in growth medium revealed that under conditions permitting growth and metabolism, toxin bound to cells was lost. The possibility that the growth medium was in-activating toxin molecules was ruled out by showing that unbound toxin molecules in the medium were fully capable of binding to fresh cultures. 相似文献
The extracellular domain of the nicotinic acetylcholine receptor isoforms formed by three α4 and two β2 subunits ((α4)3(β2)2 nAChR) harbors two high-affinity “canonical” acetylcholine (ACh)-binding sites located in the two α4:β2 intersubunit interfaces and a low-affinity “noncanonical” ACh-binding site located in the α4:α4 intersubunit interface. In this study, we used ACh, cytisine, and nicotine (which bind at both the α4:α4 and α4:β2 interfaces), TC-2559 (which binds at the α4:β2 but not at the α4:α4 interface), and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI, which binds at the α4:α4 but not at the α4:β2 interface), to investigate the binding and gating properties of CMPI at the α4:α4 interface. We recorded whole-cell currents from Xenopus laevis oocytes expressing (α4)3(β2)2 nAChR in response to applications of these ligands, alone or in combination. The electrophysiological data were analyzed in the framework of a modified Monod–Wyman–Changeux allosteric activation model. We show that CMPI is a high-affinity, high-efficacy agonist at the α4:α4 binding site and that its weak direct activating effect is accounted for by its inability to productively interact with the α4:β2 sites. The data presented here enhance our understanding of the functional contributions of ligand binding at the α4:α4 subunit interface to (α4)3(β2)2 nAChR-channel gating. These findings support the potential use of α4:α4 specific ligands to increase the efficacy of the neurotransmitter ACh in conditions associated with decline in nAChRs activity in the brain. 相似文献
Amyloid β protein (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). Point mutations within the Aβ sequence associated with familial AD (FAD) are clustered around the central hydrophobic core of Aβ. Several types of mutations within the Aβ sequence have been identified, and the ‘Arctic’ mutation (E22G) has a purely cognitive phenotype typical of AD. Previous studies have shown that the primary result of the ‘Arctic’ mutation is increased formation of Aβ protofibrils. However, the molecular mechanism underlying this effect remains unknown. Aβ42 binds to a neuronal nicotinic acetylcholine receptor subunit, neuronal acetylcholine receptor subunit alpha‐7 (CHRNA7), with high affinity and, thus, may be involved in the pathogenesis of AD. Therefore, to clarify the molecular mechanism of Arctic mutation‐mediated FAD, we focused on CHRNA7 as a target molecule of Arctic Aβ. We performed an in vitro binding assay using purified CHRNA7 and synthetic Arctic Aβ40, and demonstrated that Arctic Aβ40 specifically bound to CHRNA7. The aggregation of Arctic Aβ40 was enhanced with the addition of CHRNA7. Furthermore, the function of CHRNA7 was detected by measuring Ca2+ flux and phospho‐p44/42 MAPK (ERK1/2) activation. Our results indicated that Arctic Aβ40 aggregation was enhanced by the addition of CHRNA7, which destabilized the function of CHRNA7 via inhibition of Ca2+ responses and activation of ERK1/2. These findings indicate that Arctic Aβ mutation may be involved in the mechanism underlying FAD. This mechanism may involve binding and aggregation, leading to the inhibition of CHRNA7 functions.
A neuronal F‐box protein FSN‐1 regulates Caenorhabditis elegans neuromuscular junction development by negatively regulating DLK‐mediated MAPK signalling. In the present study, we show that attenuation of insulin/IGF signalling also contributes to FSN‐1‐dependent synaptic development and function. The aberrant synapse morphology and synaptic transmission in fsn‐1 mutants are partially and specifically rescued by reducing insulin/IGF‐signalling activity in postsynaptic muscles, as well as by reducing the activity of EGL‐3, a prohormone convertase that processes agonistic insulin/IGF ligands INS‐4 and INS‐6, in neurons. FSN‐1 interacts with, and potentiates the ubiquitination of EGL‐3 in vitro, and reduces the EGL‐3 level in vivo. We propose that FSN‐1 may negatively regulate insulin/IGF signalling, in part, through EGL‐3‐dependent insulin‐like ligand processing. 相似文献
Increases of cytokine in the blood play important roles in the pathogenesis of influenza‐associated encephalopathy. TNF‐α was administered intravenously to wild‐type mice, after which blood, CSF and brain tissue were obtained, and changes in BBB permeability, the amounts of MMP‐9 and TIMP‐1, and the localization of activated MMP were assessed. There was a significant increase in BBB permeability after 6 and 12 hr. MMP‐9 was increased after 3 hr in the brain and cerebrospinal fluid, which was earlier than in the serum. TIMP‐1 protein in the brain increased significantly after MMP‐9 had increased. Activation of MMP‐9 was observed in neurons in the cerebral cortex and hippocampus, and in vascular endothelial cells. These findings suggest that an increase in blood TNF‐α promotes activation of MMP‐9 in the brain, and may also induce an increase in permeability of the BBB. Early activation of MMP‐9 in the brain may contribute to an early onset of neurological disorders and brain edema prior to multiple organ failure in those inflammatory diseases associated with highly increased concentrations of TNF‐α in the blood, such as sepsis, burns, trauma and influenza‐associated encephalopathy. 相似文献
Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed. 相似文献