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In this article, we review contemporary evidence that GluD receptors are functional ion channels whose depolarizing currents contribute to their biological functions, akin to all other members of the ionotropic glutamate receptor (iGluR) family.
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Receptores Ionotrópicos de GlutamatoRESUMEN
Neural communication and modulation are complex processes. Ionotropic glutamate receptors (iGluRs) significantly contribute to mediating the fast-excitatory branch of neurotransmission in the mammalian brain. Kainate receptors (KARs), a subfamily of the iGluRs, act as modulators of the neuronal circuitry by playing important roles at both the post- and presynaptic sites of specific neurons. The functional tetrameric receptors are formed by two different gene families, low agonist affinity (GluK1-GluK3) and high agonist affinity (GluK4-GluK5) subunits. These receptors garnered attention in the past three decades, and since then, much work has been done to understand their localization, interactome, physiological functions, and regulation. Cloning of the receptor subunits (GluK1-GluK5) in the early 1990s led to recombinant expression of kainate receptors in heterologous systems. This facilitated understanding of the functional differences between subunit combinations, splice variants, trafficking, and drug discovery. Structural studies of individual domains and recent full-length homomeric and heteromeric kainate receptors have revealed unique functional mechanisms, which have answered several long-standing questions in the field of kainate receptor biology. In this chapter, we review the current understanding of kainate receptors and associated disorders.
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Mamíferos , Receptores de Ácido Kaínico , Animales , Mamíferos/metabolismo , Mutación , Subunidades de Proteína/metabolismo , Receptores de Ácido Kaínico/genética , Receptores de Ácido Kaínico/metabolismoRESUMEN
Fe(II) and Ni(II) paraCEST contrast agents containing the di-pyridine macrocyclic ligand 2,2',2â³-(3,7,10-triaza-1,5(2,6)-dipyridinacycloundecaphane-3,7,10-triyl)triacetamide (DETA) are reported here. Both [Fe(DETA)]2+ and [Ni(DETA)]2+ complexes were structurally characterized. Crystallographic data revealed the seven-coordinated distorted pentagonal bipyramidal geometry of the [Fe(DETA)]·(BF4)2·MeCN complex with five coordinated nitrogen atoms from the macrocyclic ring and two coordinated oxygen atoms from two amide pendant arms. The [Ni(DETA)]·Cl2·2H2O complex was six-coordinated in nature with a distorted octahedral geometry. Four coordinated nitrogen atoms were from the macrocyclic ring, and two coordinated oxygen atoms were from two amide pendant arms. [Fe(DETA)]2+ exhibited well-resolved sharp proton resonances, whereas very broad proton resonances were observed in the case of [Ni(DETA)]2+ due to the long electronic relaxation times. The CEST peaks for the [Fe(DETA)]2+ complex showed one highly downfield-shifted and intense peak at 84 ppm with another shifted but less intense peak at 28 ppm with good CEST contrast efficiency at body temperature, whereas [Ni(DETA)]2+ showed only one highly shifted intense peak at 78 ppm from the bulk water protons. Potentiometric titrations were performed to determine the protonation constants of the ligand and the thermodynamic stability constant of the [M(DETA)]2+ (M = Fe, Co, Ni, Cu, Zn) species at 25.0 °C and I = 0.15 mol·L-1 NaClO4. Metal exchange studies confirmed the stability of the complexes in acidic medium in the presence of physiologically relevant anions and an equimolar concentration of Zn(II) ions.
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Medios de Contraste , Protones , Ligandos , Medios de Contraste/química , Estructura Molecular , DEET , Cristalografía por Rayos X , Piridinas/química , Amidas/química , Compuestos Ferrosos/química , Oxígeno , Nitrógeno , AguaRESUMEN
Mycobacterium tuberculosis (Mtb) is one of the deadliest pathogens encountered by humanity. Over the decades, its characteristic membrane organization and composition have been understood. However, there is still limited structural information and mechanistic understanding of the constituent membrane proteins critical for drug discovery pipelines. Recent advances in single-particle cryo-electron microscopy and cryo-electron tomography have provided the much-needed impetus towards structure determination of several vital Mtb membrane proteins whose structures were inaccessible via X-ray crystallography and NMR. Important insights into membrane composition and organization have been gained via a combination of electron tomography and biochemical and biophysical assays. In addition, till the time of writing this review, 75 new structures of various Mtb proteins have been reported via single-particle cryo-EM. The information obtained from these structures has improved our understanding of the mechanisms of action of these proteins and the physiological pathways they are associated with. These structures have opened avenues for structure-based drug design and vaccine discovery programs that might help achieve global-TB control. This review describes the structural features of selected membrane proteins (type VII secretion systems, Rv1819c, Arabinosyltransferase, Fatty Acid Synthase, F-type ATP synthase, respiratory supercomplex, ClpP1P2 protease, ClpB disaggregase and SAM riboswitch), their involvement in physiological pathways, and possible use as a drug target. Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. The Cryo-EM and tomography have simplified the understanding of the mycobacterial membrane organization. Some proteins are located in the plasma membrane; some span the entire envelope, while some, like MspA, are located in the mycomembrane. Cryo-EM has made the study of such membrane proteins feasible.
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Proteínas de la Membrana , Mycobacterium tuberculosis , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X , Descubrimiento de Drogas/métodos , Proteínas de la Membrana/químicaRESUMEN
GluD2 receptor belongs to the orphan delta family of glutamate receptor ion channels. These receptors play key roles in synaptogenesis and synaptic plasticity and are associated with multiple neuronal disorders like schizophrenia, autism spectrum disorder, cerebellar ataxia, intellectual disability, paraplegia, retinal dystrophy, etc. Despite the importance of these receptors in CNS, insights into full-length GluD2 receptor structure is missing till-date. Here we report cryo-electron microscopy structure of the rat GluD2 receptor in the presence of calcium ions and the ligand 7-chlorokynurenic acid, elucidating its 3D architecture. The structure reveals a non-swapped architecture at the extracellular amino-terminal (ATD), and ligand-binding domain (LBD) interface similar to that observed in GluD1; however, the organization and arrangement of the ATD and LBD domains in GluD2 are unique. While our results demonstrate that non-swapped architecture is conserved in the delta receptor family, they also highlight the differences that exist between the two member receptors; GluD1 and GluD2.
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Microscopía por Crioelectrón , Neuronas/ultraestructura , Receptores de Glutamato/ultraestructura , Receptores Ionotrópicos de Glutamato/ultraestructura , Animales , Sistema Nervioso Central/patología , Sistema Nervioso Central/ultraestructura , Humanos , Ligandos , Plasticidad Neuronal/genética , Neuronas/metabolismo , Neuronas/patología , Dominios Proteicos/genética , Ratas , Receptores de Glutamato/genética , Receptores Ionotrópicos de Glutamato/genéticaRESUMEN
Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.
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Microscopía por Crioelectrón , Receptores AMPA/metabolismo , Receptores AMPA/ultraestructura , Receptores de Ácido Kaínico/metabolismo , Receptores de Ácido Kaínico/ultraestructura , Animales , Sitios de Unión , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Ácido Glutámico/farmacología , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Modelos Moleculares , Estructura Terciaria de Proteína/efectos de los fármacos , Ratas , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/química , Receptores de Ácido Kaínico/química , Receptor de Ácido Kaínico GluK2RESUMEN
X-ray crystal structures for the soluble amino-terminal and ligand-binding domains of glutamate receptor ion channels, combined with a 3.6-Å-resolution structure of the full-length AMPA receptor GluA2 homotetramer, provide unique insights into the mechanisms of the assembly and function of glutamate receptor ion channels. Increasingly sophisticated biochemical, computational, and electrophysiological experiments are beginning to reveal the mechanism of action of partial agonists and suggest new models for the mechanism of action of allosteric modulators. Newly identified NMDA receptor ligands acting at novel sites offer hope for the development of subtype-selective modulators. The many unresolved issues include the role of the amino-terminal domain in AMPA receptor signaling and the mechanisms by which auxiliary proteins regulate receptor activity. The structural basis for ion permeation and ion channel block also remain areas of uncertainty, and despite substantial progress, molecular dynamics simulations have yet to reveal how glutamate binding opens the ion channel pore.
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Canales Iónicos/fisiología , Receptores de Glutamato/fisiología , Receptores Ionotrópicos de Glutamato/química , Receptores Ionotrópicos de Glutamato/fisiología , Animales , Simulación por Computador , Cristalografía por Rayos X , Humanos , Canales Iónicos/química , Receptores AMPA/química , Receptores AMPA/fisiología , Receptores de Glutamato/química , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiologíaRESUMEN
The SMc01113/YbeY protein, belonging to the UPF0054 family, is highly conserved in nearly every bacterium. However, the function of these proteins still remains elusive. Our results show that SMc01113/YbeY proteins share structural similarities with the MID domain of the Argonaute (AGO) proteins, and might similarly bind to a small-RNA (sRNA) seed, making a special interaction with the phosphate on the 5'-side of the seed, suggesting they may form a component of the bacterial sRNA pathway. Indeed, eliminating SMc01113/YbeY expression in Sinorhizobium meliloti produces symbiotic and physiological phenotypes strikingly similar to those of the hfq mutant. Hfq, an RNA chaperone, is central to bacterial sRNA-pathway. We evaluated the expression of 13 target genes in the smc01113 and hfq mutants. Further, we predicted the sRNAs that may potentially target these genes, and evaluated the accumulation of nine sRNAs in WT and smc01113 and hfq mutants. Similar to hfq, smc01113 regulates the accumulation of sRNAs as well as the target mRNAs. AGOs are central components of the eukaryotic sRNA machinery and conceptual parallels between the prokaryotic and eukaryotic sRNA pathways have long been drawn. Our study provides the first line of evidence for such conceptual parallels. Furthermore, our investigation gives insights into the sRNA-mediated regulation of stress adaptation in S. meliloti.
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Proteínas Bacterianas/fisiología , ARN Pequeño no Traducido/metabolismo , Sinorhizobium meliloti/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/genética , Proteína de Factor 1 del Huésped/fisiología , Datos de Secuencia Molecular , Mutación , Fenotipo , Complejo Silenciador Inducido por ARN/química , Alineación de Secuencia , Sinorhizobium meliloti/metabolismo , SimbiosisRESUMEN
Glutamate receptor ion channels (iGluRs) are excitatory neurotransmitter receptors with a unique molecular architecture in which the extracellular domains assemble as a dimer of dimers. The structure of individual dimer assemblies has been established previously for both the isolated ligand-binding domain (LBD) and more recently for the larger amino terminal domain (ATD). How these dimers pack to form tetrameric assemblies in intact iGluRs has remained controversial. Using recently solved crystal structures for the GluK2 kainate receptor ATD as a guide, we performed cysteine mutant cross-linking experiments in full-length tetrameric GluK2 to establish how the ATD packs in a dimer of dimers assembly. A similar approach, using a full-length AMPA receptor GluA2 crystal structure as a guide, was used to design cysteine mutant cross-links for the GluK2 LBD dimer of dimers assembly. The formation of cross-linked tetramers in full-length GluK2 by combinations of ATD and LBD mutants which individually produce only cross-linked dimers suggests that subunits in the ATD and LBD layers swap dimer partners. Functional studies reveal that cross-linking either the ATD or the LBD inhibits activation of GluK2 and that, in the LBD, cross-links within and between dimers have different effects. These results establish that kainate and AMPA receptors have a conserved extracellular architecture and provide insight into the role of individual dimer assemblies in activation of ion channel gating.
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Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Humanos , Activación del Canal Iónico , Ligandos , Modelos Moleculares , Mutación , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ratas , Receptores de Ácido Kaínico/genética , Receptor de Ácido Kaínico GluK2RESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) is known for Warburg effect and defects in the mitochondria. AMP-dependent kinase (AMPK) activates the downstream transcription factors PGC-1α, PGC-1ß, or FOXO1, which participate in mitochondrial biogenesis. 5- aminoimidazole-4-carboxamide riboside (AICAR) is an analog of adenosine monophosphate and is a direct activator of AMPK. OBJECTIVES: In the present study, we have made an attempt to understand the influence of AICAR on TNBC cells, MDA-MB-231, and the underlying changes in mitochondrial biogenesis, if any. METHODS: We investigated AICAR induced changes in cell viability, apoptosis, migratory potential, and changes in the sensitivity of doxorubicin. RESULTS: In response to the treatment of MDA-MB-231 breast cancer cells with 750 µM of AICAR for 72 hours, followed by 48 hours in fresh media without AICAR, we observed a decrease in viability via MTT assay, reduction in cell numbers along with the apoptotic appearance, increased cell death by ELISA, decreased lactate in conditioned medium and decrease in migration by scratch and transwell migration assays. These changes in the cancer phenotype were accompanied by an increase in mitochondrial biogenesis, as observed by increased mitochondrial DNA to nuclear DNA ratio, a decrease in lactic acid concentration, an increase in MitoTracker green and red staining, and increased expression of transcription factors PGC-1α, NRF-1, NRF-2, and TFAM, contributing to mitochondrial biogenesis. Pre-treatment of cells with AICAR for 72 hours followed by 48 hours treatment with 1 µM doxorubicin showed an increased sensitivity to doxorubicin as assessed by the MTT assay. CONCLUSION: Our results show that AICAR exerts beneficial effects on TNBC cells, possibly via switching off the Warburg effect and switching on the anti-Warburg effect through mitochondrial modulation.
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Neoplasias de la Mama Triple Negativas , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Doxorrubicina/farmacología , Humanos , Imidazoles , Mitocondrias , Ribonucleótidos , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
Glutamate delta-1 (GluD1) and delta-2 (GluD2) receptors belong to the orphan GluD subfamily of ionotropic glutamate receptors (iGluRs). GluDs were classified as ionotropic glutamate receptors based on their sequence similarity. Two decades after these GluDs were first cloned they are still considered "orphan" due to a lack of knowledge of the endogenous ligands that can activate them. Nevertheless, they are crucial for synapse formation, maturation and maintenance of CNS functions, and are implicated in multiple neuronal disorders, including schizophrenia, autism spectrum disorder and depressive disorders. Over the last decade significant discoveries have been made, include role of GluD receptors in mediating trans-synaptic interactions and their unique non-swapped architecture, which is distinct from other ionotropic glutamate receptors. Also, the prospect of GluD ionotropic activity being regulated by direct interaction with metabotropic glutamate receptors is exciting. These discoveries will likely drive the field in the future, providing direction to GluD research. LINKED ARTICLES: This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.
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Trastorno del Espectro Autista , Receptores de Glutamato Metabotrópico , Glutamatos , Humanos , Receptores Ionotrópicos de Glutamato , Receptores de Glutamato Metabotrópico/metabolismo , Receptores Opioides delta/metabolismoRESUMEN
Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis. Despite decades of research driving advancements in drug development and discovery against TB, it still leads among the causes of deaths due to infectious diseases. We are yet to develop an effective treatment course or a vaccine that could help us eradicate TB. Some key issues being prolonged treatment courses, inadequate drug intake, and the high dropout rate of patients during the treatment course. Hence, we require drugs that could accelerate the elimination of bacteria, shortening the treatment duration. It is high time we evaluate the probable lacunas in research holding us back in coming up with a treatment regime and/or a vaccine that would help control TB spread. Years of dedicated and focused research provide us with a lead molecule that goes through several tests, trials, and modifications to transform into a 'drug'. The transformation from lead molecule to 'drug' is governed by several factors determining its success or failure. In the present review, we have discussed drugs that are part of the currently approved treatment regimen, their limitations, vaccine candidates under trials, and current issues in research that need to be addressed. While we are waiting for the path-breaking treatment for TB, these factors should be considered during the ongoing quest for novel yet effective anti-tubercular. If these issues are addressed, we could hope to develop a more effective treatment that would cure multi/extremely drug-resistant TB and help us meet the WHO's targets for controlling the global TB pandemic within the prescribed timeline.
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Neural networks with precise connection are compulsory for learning and memory. Various cellular events occur during the genesis of dendritic spines to their maturation, synapse formation, stabilization of the synapse, and proper signal transmission. The cortical actin cytoskeleton and its multiple regulatory proteins are crucial for the above cellular events. The different types of ionotropic glutamate receptors (iGluRs) present on the postsynaptic density (PSD) are also essential for learning and memory. Interaction of the iGluRs in association of their auxiliary proteins with actin cytoskeleton regulated by actin-binding proteins (ABPs) are required for precise long-term potentiation (LTP) and long-term depression (LTD). There has been a quest to understand the mechanistic detail of synapse function involving these receptors with dynamic actin cytoskeleton. A major, emerging area of investigation is the relationship between ABPs and iGluRs in synapse development. In this review we have summarized the current understanding of iGluRs functioning with respect to the actin cytoskeleton, scaffolding proteins, and their regulators. The AMPA, NMDA, Delta and Kainate receptors need the stable underlying actin cytoskeleton to anchor through synaptic proteins for precise synapse formation. The different types of ABPs present in neurons play a critical role in dynamizing/stabilizing the actin cytoskeleton needed for iGluRs function.
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Enigmatic orphan glutamate delta receptors (GluD) are one of the four classes of the ionotropic glutamate receptors (iGluRs) that play key roles in synaptic transmission and plasticity. While members of other iGluR families viz AMPA, NMDA, and kainate receptors are gated by glutamate, the GluD receptors neither bind glutamate nor evoke ligand-induced currents upon binding of glycine and D-serine. Thus, the GluD receptors were considered to function as structural proteins that facilitate the formation, maturation, and maintenance of synapses in the hippocampus and cerebellum. Recent work has revealed that GluD receptors have extensive crosstalk with metabotropic glutamate receptors (mGlus) and are also gated by their activation. The latest development of a novel optopharamcological tool and the cryoEM structures of GluD receptors would help define the molecular and chemical basis of the GluD receptor's role in synaptic physiology. This article is part of the special Issue on "Glutamate Receptors - Orphan iGluRs".
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Receptores de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Cerebelo , Microscopía por Crioelectrón , Hipocampo , Humanos , Plasticidad Neuronal/fisiología , Receptores de Glutamato/fisiología , Receptores de Glutamato/ultraestructura , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores Ionotrópicos de Glutamato/fisiología , Receptores Ionotrópicos de Glutamato/ultraestructura , Receptores de Glutamato Metabotrópico/fisiología , Relación Estructura-ActividadRESUMEN
Cryo-electron microscopy (CryoEM) has superseded X-ray crystallography and NMR to emerge as a popular and effective tool for structure determination in recent times. It has become indispensable for the characterization of large macromolecular assemblies, membrane proteins, or samples that are limited, conformationally heterogeneous, and recalcitrant to crystallization. Besides, it is the only tool capable of elucidating high-resolution structures of macromolecules and biological assemblies in situ. A state-of-the-art electron microscope operable at cryo-temperature helps preserve high-resolution details of the biological sample. The structures can be determined, either in isolation via single-particle analysis (SPA) or helical reconstruction, electron diffraction (ED) or within the cellular environment via cryo-electron tomography (cryoET). All the three streams of SPA, ED, and cryoET (along with subtomogram averaging) have undergone significant advancements in recent times. This has resulted in breaking the boundaries with respect to both the size of the macromolecules/assemblies whose structures could be determined along with the visualization of atomic details at resolutions unprecedented for cryoEM. In addition, the collection of larger datasets combined with the ability to sort and process multiple conformational states from the same sample are providing the much-needed link between the protein structures and their functions. In overview, these developments are helping scientists decipher the molecular mechanism of critical cellular processes, solve structures of macromolecules that were challenging targets for structure determination until now, propelling forward the fields of biology and biomedicine. Here, we summarize recent advances and key contributions of the three cryo-electron microscopy streams of SPA, ED, and cryoET.
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Disciplinas de las Ciencias Biológicas , Tomografía con Microscopio Electrónico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Sustancias MacromolecularesRESUMEN
Kainate receptors play fundamental roles in regulating synaptic transmission and plasticity in central nervous system and are regulated by their cognate auxiliary subunits Neuropilin and tolloid-like proteins 1 and 2 (Neto). While electrophysiology-based insights into functions of Neto proteins are known, biophysical and biochemical studies into Neto proteins have been largely missing till-date. Our biochemical, biophysical, and functional characterization of the purified extracellular domain (ECD) of Neto1 shows that Neto1-ECD exists as monomers in solution and has a micromolar affinity for GluK2 receptors in apo state or closed state. Remarkably, the affinity was ~2.8 fold lower for receptors trapped in the desensitized state, highlighting the conformation-dependent interaction of Neto proteins with kainate receptors. SAXS analysis of Neto1-ECD reveals that their dimensions are long enough to span the entire extracellular domain of kainate receptors. The shape and conformation of Neto1-ECD seems to be altered by calcium ions pointing towards its possible role in modulating Neto1 functions. Functional assays using GluK2 receptors and GluK2/GluA2 chimeric receptors reveal a differential role of Neto1 domains in modulating receptor functions. Although the desensitization rate was not affected by the Neto1-ECD, the recovery rates from the desensitized state are altered.
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Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Dominios y Motivos de Interacción de Proteínas , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Animales , Fenómenos Químicos , Fenómenos Electrofisiológicos , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Técnicas de Placa-Clamp , Unión Proteica , Ratas , Proteínas Recombinantes/químicaRESUMEN
Particle identification and selection, which is a prerequisite for high-resolution structure determination of biological macromolecules via single-particle cryo-electron microscopy poses a major bottleneck for automating the steps of structure determination. Here, we present a generalized deep learning tool, CASSPER, for the automated detection and isolation of protein particles in transmission microscope images. This deep learning tool uses Semantic Segmentation and a collection of visually prepared training samples to capture the differences in the transmission intensities of protein, ice, carbon, and other impurities found in the micrograph. CASSPER is a semantic segmentation based method that does pixel-level classification and completely eliminates the need for manual particle picking. Integration of Contrast Limited Adaptive Histogram Equalization (CLAHE) in CASSPER enables high-fidelity particle detection in micrographs with variable ice thickness and contrast. A generalized CASSPER model works with high efficiency on unseen datasets and can potentially pick particles on-the-fly, enabling data processing automation.
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Microscopía por Crioelectrón , Aprendizaje Profundo , Procesamiento de Imagen Asistido por Computador , Proteínas/ultraestructura , Imagen Individual de Molécula , Animales , Humanos , Modelos Moleculares , Conformación Proteica , SemánticaRESUMEN
Ionotropic orphan delta (GluD) receptors are not gated by glutamate or any other endogenous ligand but are grouped with ionotropic glutamate receptors (iGluRs) based on sequence similarity. GluD1 receptors play critical roles in synaptogenesis and synapse maintenance and have been implicated in neuronal disorders, including schizophrenia, cognitive deficits, and cerebral ataxia. Here we report cryo-EM structures of the rat GluD1 receptor complexed with calcium and the ligand 7-chlorokynurenic acid (7-CKA), elucidating molecular architecture and principles of receptor assembly. The structures reveal a non-swapped architecture at the interface of the extracellular amino-terminal domain (ATD) and the ligand-binding domain (LBD). This finding is in contrast with structures of other families of iGluRs, where the dimer partners between the ATD and LBD layers are swapped. Our results demonstrate that principles of architecture and symmetry are not conserved between delta receptors and other iGluRs and provide a molecular blueprint for understanding the functions of the 'orphan' class of iGluRs.
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Multimerización de Proteína , Receptores Ionotrópicos de Glutamato/química , Animales , Calcio/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Ácido Quinurénico/análogos & derivados , Ácido Quinurénico/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , RatasRESUMEN
Kainate receptors belong to the ionotropic glutamate receptor family and play critical roles in the regulation of synaptic networks. The kainate receptor subunit GluK3 has unique functional properties and contributes to presynaptic facilitation at the hippocampal mossy fiber synapses along with roles at the post-synapses. To gain structural insights into the unique functional properties and dynamics of GluK3 receptor, we imaged them via electron microscopy in the apo-state and in complex with either agonist kainate or antagonist UBP301. Our analysis of all the GluK3 full-length structures not only provides insights into the receptor transitions between desensitized and closed states but also reveals a "non-classical" conformation of neurotransmitter binding domain in the closed-state distinct from that observed in AMPA and other kainate receptor structures. We show by molecular dynamics simulations that Asp759 influences the stability of the LBD dimers and hence could be responsible for the observed conformational variability and dynamics of the GluK3 via electron microscopy. Lower dimer stability could explain faster desensitization and low agonist sensitivity of GluK3. In overview, our work helps to associate biochemistry and physiology of GluK3 receptors with their structural biology and offers structural insights into the unique functional properties of these atypical receptors.
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Microscopía por Crioelectrón , Neurotransmisores/metabolismo , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/ultraestructura , Ácido Aspártico/química , Células HEK293 , Humanos , Ácido Kaínico/metabolismo , Ligandos , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Receptores de Ácido Kaínico/aislamiento & purificación , Receptor Kainato GluK3RESUMEN
GluK3-kainate receptors are atypical members of the iGluR family that reside at both the pre- and postsynapse and play a vital role in the regulation of synaptic transmission. For a better understanding of structural changes that underlie receptor functions, GluK3 receptors were trapped in desensitized and resting/closed states and structures analyzed using single particle cryo-electron microscopy. While the desensitized GluK3 has domain organization as seen earlier for another kainate receptor-GluK2, antagonist bound GluK3 trapped a resting state with only two LBD domains in dimeric arrangement necessary for receptor activation. Using structures as a guide, we show that the N-linked glycans at the interface of GluK3 ATD and LBD likely mediate inter-domain interactions and attune receptor-gating properties. The mutational analysis also identified putative N-glycan interacting residues. Our results provide a molecular framework for understanding gating properties unique to GluK3 and exploring the role of N-linked glycosylation in their modulation.