RESUMO
Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.
Assuntos
Encéfalo , Microglia , Axônios , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Macrófagos/fisiologia , Microglia/patologia , MorfogêneseRESUMO
Delta receptors (GluD1 and GluD2), members of the large ionotropic glutamate receptor (iGluR) family, play a central role in numerous neurodevelopmental and psychiatric disorders. The amino-terminal domain (ATD) of GluD orchestrates synapse formation and maturation processes through its interaction with the Cbln family of synaptic organizers and neurexin (Nrxn). The transsynaptic triad of Nrxn-Cbln-GluD also serves as a potent regulator of synaptic plasticity, at both excitatory and inhibitory synapses. Despite these recognized functions, there is still debate as to whether GluD functions as a "canonical" ion channel, similar to other iGluRs. A recent report proposes that the ATD of GluD2 imposes conformational constraints on channel activity; removal of this constraint by binding to Cbln1 and Nrxn, or removal of the ATD, reveals channel activity in GluD2 upon administration of glycine (Gly) and d-serine (d-Ser), two GluD ligands. We were able to reproduce currents when Gly or d-Ser was administered to clusters of heterologous human embryonic kidney 293 (HEK293) cells expressing Cbln1, GluD2 (or GluD1), and Nrxn. However, Gly or d-Ser, but also l-glutamate (l-Glu), evoked similar currents in naive (i.e., untransfected) HEK293 cells and in GluD2-null Purkinje neurons. Furthermore, no current was detected in isolated HEK293 cells expressing GluD2 lacking the ATD upon administration of Gly. Taken together, these results cast doubt on the previously proposed hypothesis that extracellular ligands directly gate wild-type GluD channels.
Assuntos
Ativação do Canal Iônico , Receptores de Glutamato , Animais , Humanos , Camundongos , Glicina/metabolismo , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Ligantes , Receptores de Glutamato/metabolismo , Serina/metabolismoRESUMO
Light-controllable tools provide powerful means to manipulate and interrogate brain function with relatively low invasiveness and high spatiotemporal precision. Although optogenetic approaches permit neuronal excitation or inhibition at the network level, other technologies, such as optopharmacology (also known as photopharmacology) have emerged that provide molecular-level control by endowing light sensitivity to endogenous biomolecules. In this Review, we discuss the challenges and opportunities of photocontrolling native neuronal signalling pathways, focusing on ion channels and neurotransmitter receptors. We describe existing strategies for rendering receptors and channels light sensitive and provide an overview of the neuroscientific insights gained from such approaches. At the crossroads of chemistry, protein engineering and neuroscience, optopharmacology offers great potential for understanding the molecular basis of brain function and behaviour, with promises for future therapeutics.
Assuntos
Canais Iônicos/metabolismo , Neurônios/metabolismo , Optogenética/tendências , Processos Fotoquímicos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Moduladores de Transporte de Membrana/farmacologia , Neurônios/química , Neurônios/efeitos dos fármacos , Optogenética/métodos , Processos Fotoquímicos/efeitos dos fármacos , Receptores Acoplados a Proteínas G/químicaRESUMO
NMDA receptors (NMDARs) are glutamate-gated ion channels that are key mediators of excitatory neurotransmission and synaptic plasticity throughout the central nervous system. They form massive heterotetrameric complexes endowed with unique allosteric capacity provided by eight extracellular clamshell-like domains arranged as two superimposed layers. Despite an increasing number of full-length NMDAR structures, how these domains cooperate in an intact receptor to control its activity remains poorly understood. Here, combining single-molecule and macroscopic electrophysiological recordings, cysteine biochemistry, and in silico analysis, we identify a rolling motion at a yet unexplored interface between the two constitute dimers in the agonist-binding domain (ABD) layer as a key structural determinant in NMDAR activation and allosteric modulation. This rotation acts as a gating switch that tunes channel opening depending on the conformation of the membrane-distal N-terminal domain (NTD) layer. Remarkably, receptors locked in a rolled state display "super-activity" and resistance to NTD-mediated allosteric modulators. Our work unveils how NMDAR domains move in a concerted manner to transduce long-range conformational changes between layers and command receptor channel activity.
Assuntos
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Animais , Simulação por Computador , Cisteína/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais , Imagem Individual de Molécula , Xenopus laevisRESUMO
Extensive evidence links Glutamate receptor, ionotropic, NMDA2B (GRIN2B), encoding the GluN2B/NR2B subunit of N-methyl-D-aspartate receptors (NMDARs), with various neurodevelopmental disorders, including autism spectrum disorders (ASDs), but the underlying mechanisms remain unclear. In addition, it remains unknown whether mutations in GluN2B, which starts to be expressed early in development, induces early pathophysiology that can be corrected by early treatments for long-lasting effects. We generated and characterized Grin2b-mutant mice that carry a heterozygous, ASD-risk C456Y mutation (Grin2b+/C456Y). In Grin2b+/C456Y mice, GluN2B protein levels were strongly reduced in association with decreased hippocampal NMDAR currents and NMDAR-dependent long-term depression (LTD) but unaltered long-term potentiation, indicative of mutation-induced protein degradation and LTD sensitivity. Behaviorally, Grin2b+/C456Y mice showed normal social interaction but exhibited abnormal anxiolytic-like behavior. Importantly, early, but not late, treatment of young Grin2b+/C456Y mice with the NMDAR agonist D-cycloserine rescued NMDAR currents and LTD in juvenile mice and improved anxiolytic-like behavior in adult mice. Therefore, GluN2B-C456Y haploinsufficiency decreases GluN2B protein levels, NMDAR-dependent LTD, and anxiety-like behavior, and early activation of NMDAR function has long-lasting effects on adult mouse behavior.
Assuntos
Ansiedade/genética , Hipocampo/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Animais , Ansiedade/fisiopatologia , Comportamento Animal/efeitos dos fármacos , Ciclosserina/farmacologia , Potenciais Pós-Sinápticos Excitadores/genética , Técnicas de Introdução de Genes , Haploinsuficiência/genética , Heterozigoto , Hipocampo/metabolismo , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos Mutantes , Mutação , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Approaches to remotely control and monitor ion channel operation with light are expanding rapidly in the biophysics and neuroscience fields. A recent development directly introduces light sensitivity into proteins by utilizing photosensitive unnatural amino acids (UAAs) incorporated using the genetic code expansion technique. The introduction of UAAs results in unique molecular level control and, when combined with the maximal spatiotemporal resolution and poor invasiveness of light, enables direct manipulation and interrogation of ion channel functionality. Here, we review the diverse applications of light-sensitive UAAs in two superfamilies of ion channels (voltage- and ligand-gated ion channels; VGICs and LGICs) and summarize existing UAA tools, their mode of action, potential, caveats, and technical considerations to their use in illuminating ion channel structure and function.
Assuntos
Aminoácidos/metabolismo , Aminoácidos/efeitos da radiação , Canais Iônicos/química , Canais Iônicos/metabolismo , Luz , Animais , Canais Iônicos/efeitos da radiaçãoRESUMO
NMDA receptors (NMDARs) are glutamate-gated ion channels that play key roles in synaptic transmission and plasticity. Both hyper- and hypo-activation of NMDARs are deleterious to neuronal function. In particular, NMDAR hypofunction is involved in a wide range of neurological and psychiatric conditions like schizophrenia, intellectual disability, age-dependent cognitive decline, or Alzheimer's disease. While early medicinal chemistry efforts were mostly focused on the development of NMDAR antagonists, the last 10 years have seen a boom in the development of NMDAR positive allosteric modulators (PAMs). Here we review the currently developed NMDAR PAMs, their pharmacological profiles and mechanisms of action, as well as their physiological effects in healthy animals and animal models of NMDAR hypofunction. In light of the complexity of physiological outcomes of NMDAR PAMs in vivo, we discuss the remaining challenges and questions that need to be addressed to better grasp and predict the therapeutic potential of NMDAR positive allosteric modulation.
Assuntos
Doença de Alzheimer , Receptores de N-Metil-D-Aspartato , Regulação Alostérica , Animais , Ácido Glutâmico , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão SinápticaRESUMO
Ionotropic glutamate receptors (iGluRs) are a major class of ligand-gated ion channels that are widespread in the living kingdom. Their critical role in excitatory neurotransmission and brain function of arthropods and vertebrates has made them a compelling subject of interest for neurophysiologists and pharmacologists. This is particularly true for NMDA receptor (NMDARs), a subclass of iGluRs that act as central drivers of synaptic plasticity in the CNS. How and when the unique properties of NMDARs arose during evolution, and how they relate to the evolution of the nervous system, remain open questions. Recent years have witnessed a boom in both genomic and structural data, such that it is now possible to analyse the evolution of iGluR genes on an unprecedented scale and within a solid molecular framework. In this review, combining insights from phylogeny, atomic structure and physiological and mechanistic data, we discuss how evolution of NMDAR motifs and sequences shaped their architecture and functionalities. We trace differences and commonalities between NMDARs and other iGluRs, emphasizing a few distinctive properties of the former regarding ligand binding and gating, permeation, allosteric modulation and intracellular signalling. Finally, we speculate on how specific molecular properties of iGuRs arose to supply new functions to the evolving structure of the nervous system, from early metazoan to present mammals.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores de N-Metil-D-Aspartato , Animais , Fenômenos Biofísicos , Receptores Ionotrópicos de Glutamato/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão SinápticaRESUMO
Hyperactivity of glutamatergic corticostrial pathways is recognized as a key pathophysiological mechanism contributing to development of PD symptoms and dopaminergic neurotoxicity. Subset of corticostriatal projection neurons uses Zn2+ as a co-transmitter alongside glutamate, but the role of synaptically released Zn2+ in PD remains unexplored. We used genetically modified mice and pharmacological tools in combination with 6-hydroxydopamine (6-OHDA) lesion models of PD to investigate the contribution of synaptic zinc to disease associated behavioral deficits and neurodegeneration. Vesicular zinc transporter-3 (ZnT3) knockout mice lacking releasable Zn2+ were more resistant to locomotor deficit and memory impairment of nigrostriatal dopamine (DA) denervation compared to wildtype littermates. The loss of striatal dopaminergic fibers was comparable between genotypes, indicating that synaptically released Zn2+ contributes to behavioral deficits but not neurotoxic effects of 6-OHDA. To gain further insight into the mechanisms of Zn2+ actions, we used the extracellular Zn2+ chelator CaEDTA and knock-in mice lacking the high affinity Zn2+ inhibition of GluN2A-containing NMDA receptors (GluN2A-NMDARs). Acute chelation of extracellular Zn2+ in the striatum restored locomotor deficit of 6-OHDA lesion, confirming that synaptic Zn2+ suppresses locomotor behavior. Disruption of the Zn2+-GluN2A interaction had, on the other hand, no impact on locomotor deficit or neurotoxic effect of 6-OHDA. Collectively, these findings provide clear evidence for the implication of striatal synaptic Zn2+ in the pathophysiology of PD. They unveil that synaptic Zn2+ plays predominantly a detrimental role by promoting motor and cognitive deficits caused by nigrostriatal DA denervation, pointing towards new therapeutic interventions.
Assuntos
Cognição , Locomoção , Doença de Parkinson/metabolismo , Doença de Parkinson/psicologia , Zinco/metabolismo , Animais , Comportamento Animal , Proteínas de Transporte de Cátions/genética , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Masculino , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxidopamina/administração & dosagem , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/psicologia , Vesículas Sinápticas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
NMDA receptors (NMDARs) are glutamate-gated ion channels and are crucial for neuronal communication. NMDARs form tetrameric complexes that consist of several homologous subunits. The subunit composition of NMDARs is plastic, resulting in a large number of receptor subtypes. As each receptor subtype has distinct biophysical, pharmacological and signalling properties, there is great interest in determining whether individual subtypes carry out specific functions in the CNS in both normal and pathological conditions. Here, we review the effects of subunit composition on NMDAR properties, synaptic plasticity and cellular mechanisms implicated in neuropsychiatric disorders. Understanding the rules and roles of NMDAR diversity could provide new therapeutic strategies against dysfunctions of glutamatergic transmission.
Assuntos
Transtornos Mentais/fisiopatologia , Plasticidade Neuronal/fisiologia , Subunidades Proteicas/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Humanos , Modelos Biológicos , Transdução de Sinais/fisiologiaRESUMO
Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.
Assuntos
Luz , Engenharia de Proteínas , Subunidades Proteicas/metabolismo , Receptores Ionotrópicos de Glutamato/genética , Animais , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos da radiação , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos da radiação , Estrutura Terciária de Proteína , Ratos , Receptores Ionotrópicos de Glutamato/química , Raios Ultravioleta , XenopusRESUMO
N-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels that play key roles in brain physiology and pathology. Because numerous pathologic conditions involve NMDAR overactivation, subunit-selective antagonists hold strong therapeutic potential, although clinical successes remain limited. Among the most promising NMDAR-targeting drugs are allosteric inhibitors of GluN2B-containing receptors. Since the discovery of ifenprodil, a range of GluN2B-selective compounds with strikingly different structural motifs have been identified. This molecular diversity raises the possibility of distinct binding sites, although supporting data are lacking. Using X-ray crystallography, we show that EVT-101, a GluN2B antagonist structurally unrelated to the classic phenylethanolamine pharmacophore, binds at the same GluN1/GluN2B dimer interface as ifenprodil but adopts a remarkably different binding mode involving a distinct subcavity and receptor interactions. Mutagenesis experiments demonstrate that this novel binding site is physiologically relevant. Moreover, in silico docking unveils that GluN2B-selective antagonists broadly divide into two distinct classes according to binding pose. These data widen the allosteric and pharmacological landscape of NMDARs and offer a renewed structural framework for designing next-generation GluN2B antagonists with therapeutic value for brain disorders.
Assuntos
Antagonistas Adrenérgicos alfa/metabolismo , Drogas em Investigação/metabolismo , Moduladores de Transporte de Membrana/metabolismo , Modelos Moleculares , Receptores de N-Metil-D-Aspartato/metabolismo , Antagonistas Adrenérgicos alfa/química , Antagonistas Adrenérgicos alfa/farmacologia , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Biologia Computacional , Drogas em Investigação/química , Drogas em Investigação/farmacologia , Sistemas Inteligentes , Humanos , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Ligantes , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Mutação , Oxidiazóis/química , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Piridazinas/química , Piridazinas/metabolismo , Piridazinas/farmacologia , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
Ligand-gated ion channels (LGICs) mediate fast synaptic transmission in the CNS. Typically, these membrane proteins are multimeric complexes associating several homologous subunits around a central pore. Because of the large repertoire of subunits within each family, LGICs exist in vivo as multiple subtypes that differ in subunit composition and functional properties. Establishing the specific properties of individual receptor subtypes remains a major goal in the field of neuroscience and molecular pharmacology. However, isolating specific receptor subtype in recombinant systems can be problematic because of the mixture of receptor populations. This is the case for NMDA receptors (NMDARs), a large family of tetrameric glutamate-gated ion channels that play key roles in brain physiology and pathology. A significant fraction of native NMDARs are triheteromers composed of two GluN1 subunits and two different GluN2 subunits (GluN2A-D). We developed a method based on dual retention signals adapted from G-protein-coupled GABA-B receptors allowing exclusive cell surface expression of triheteromeric rat NMDARs while coexpressed diheteromeric receptors (which contain a single type of GluN2 subunit) are retained intracellularly. Using this approach, we determined the functional properties of GluN1/GluN2A/GluN2B triheteromers, one of the most abundant NMDAR subtypes in the adult forebrain, revealing their unique gating and pharmacological attributes. We envision applicability of the retention signal approach for the study of a variety of heteromeric glutamate-gated ion channel receptors with defined subunit composition.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Feminino , Ácido Glutâmico/farmacologia , Glicina/farmacologia , Dados de Sequência Molecular , Subunidades Proteicas/agonistas , Subunidades Proteicas/fisiologia , Ratos , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.
Assuntos
Poliaminas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Subunidades Proteicas/metabolismo , Alinhamento de SequênciaRESUMO
N-methyl-d-aspartate (NMDA) receptors (NMDARs) are a major class of excitatory neurotransmitter receptors in the central nervous system. They form glutamate-gated ion channels that are highly permeable to calcium and mediate activity-dependent synaptic plasticity. NMDAR dysfunction is implicated in multiple brain disorders, including stroke, chronic pain and schizophrenia. NMDARs exist as multiple subtypes with distinct pharmacological and biophysical properties that are largely determined by the type of NR2 subunit (NR2A to NR2D) incorporated in the heteromeric NR1/NR2 complex. A fundamental difference between NMDAR subtypes is their channel maximal open probability (P(o)), which spans a 50-fold range from about 0.5 for NR2A-containing receptors to about 0.01 for receptors containing NR2C and NR2D; NR2B-containing receptors have an intermediate value (about 0.1). These differences in P(o) confer unique charge transfer capacities and signalling properties on each receptor subtype. The molecular basis for this profound difference in activity between NMDAR subtypes is unknown. Here we show that the subunit-specific gating of NMDARs is controlled by the region formed by the NR2 amino-terminal domain (NTD), an extracellular clamshell-like domain previously shown to bind allosteric inhibitors, and the short linker connecting the NTD to the agonist-binding domain (ABD). The subtype specificity of NMDAR P(o) largely reflects differences in the spontaneous (ligand-independent) equilibrium between open-cleft and closed-cleft conformations of the NR2-NTD. This NTD-driven gating control also affects pharmacological properties by setting the sensitivity to the endogenous inhibitors zinc and protons. Our results provide a proof of concept for a drug-based bidirectional control of NMDAR activity by using molecules acting either as NR2-NTD 'closers' or 'openers' promoting receptor inhibition or potentiation, respectively.
Assuntos
Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Linhagem Celular , Humanos , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Prótons , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes/metabolismo , Zinco/farmacologiaRESUMO
NMDA receptors are widely expressed in the central nervous system and play a major role in excitatory synaptic transmission and plasticity. Here, we used atomic force microscopy (AFM) imaging to visualize activation-induced structural changes in the GluN1/GluN2A NMDA receptor reconstituted into a lipid bilayer. In the absence of agonist, AFM imaging revealed two populations of particles with heights above the bilayer surface of 8.6 and 3.4 nm. The taller, but not the shorter, particles could be specifically decorated by an anti-GluN1 antibody, which recognizes the S2 segment of the agonist-binding domain, indicating that the two populations represent the extracellular and intracellular regions of the receptor, respectively. In the presence of glycine and glutamate, there was a reduction in the height of the extracellular region to 7.3 nm. In contrast, the height of the intracellular domain was unaffected. Fast-scan AFM imaging combined with UV photolysis of caged glutamate permitted the detection of a rapid reduction in the height of individual NMDA receptors. The reduction in height did not occur in the absence of the co-agonist glycine or in the presence of the selective NMDA receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid, indicating that the observed structural change was caused by receptor activation. These results represent the first demonstration of an activation-induced effect on the structure of the NMDA receptor at the single-molecule level. A change in receptor size following activation could have important functional implications, in particular by affecting interactions between the NMDA receptor and its extracellular synaptic partners.
Assuntos
Microscopia de Força Atômica/métodos , Receptores de N-Metil-D-Aspartato/metabolismo , Células HEK293 , Humanos , Conformação Proteica , Receptores de N-Metil-D-Aspartato/químicaRESUMO
The past few years have witnessed dramatic progress on all frontiers of zinc neurobiology. The recent development of powerful tools, including zinc-sensitive fluorescent probes, selective chelators and genetically modified animal models, has brought a deeper understanding of the roles of this cation as a crucial intra- and intercellular signalling ion of the CNS, and hence of the neurophysiological importance of zinc-dependent pathways and the injurious effects of zinc dyshomeostasis. The development of some innovative therapeutic strategies is aimed at controlling and preventing the damaging effects of this cation in neurological conditions such as stroke and Alzheimer's disease.
Assuntos
Doenças do Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/patologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiologia , Zinco/fisiologia , Animais , Cátions Bivalentes/efeitos adversos , Cátions Bivalentes/farmacologia , Morte Celular/fisiologia , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Doenças do Sistema Nervoso Central/fisiopatologia , Humanos , Transdução de Sinais/fisiologia , Zinco/efeitos adversos , Zinco/metabolismoRESUMO
Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.