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1.
bioRxiv ; 2023 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-37577549

RESUMO

Behavioral time scale plasticity (BTSP), is a form of non-Hebbian plasticity induced by integrating pre- and postsynaptic components separated by behavioral time scale (seconds). BTSP in the hippocampal CA1 neurons underlies place cell formation. However, the molecular mechanisms underlying this behavioral time scale (eligibility trace) and synapse specificity are unknown. CaMKII can be activated in a synapse-specific manner and remain active for a few seconds, making it a compelling candidate for the eligibility trace during BTSP. Here, we show that BTSP can be induced in a single dendritic spine using 2-photon glutamate uncaging paired with postsynaptic current injection temporally separated by behavioral time scale. Using an improved CaMKII sensor, we saw no detectable CaMKII activation during this BTSP induction. Instead, we observed a dendritic, delayed, and stochastic CaMKII activation (DDSC) associated with Ca 2+ influx and plateau 20-40 s after BTSP induction. DDSC requires both pre-and postsynaptic activity, suggesting that CaMKII can integrate these two signals. Also, optogenetically blocking CaMKII 30 s after the BTSP protocol inhibited synaptic potentiation, indicating that DDSC is an essential mechanism of BTSP. IP3-dependent intracellular Ca 2+ release facilitates both DDSC and BTSP. Thus, our study suggests that the non-synapse specific CaMKII activation provides an instructive signal with an extensive time window over tens of seconds during BTSP.

2.
Mol Cell ; 82(23): 4548-4563.e4, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36309015

RESUMO

Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders.


Assuntos
Ácido Glutâmico , Receptores de N-Metil-D-Aspartato , Humanos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Transmissão Sináptica , Subunidades Proteicas/metabolismo
3.
Nat Commun ; 13(1): 923, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177668

RESUMO

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.


Assuntos
Anticorpos Monoclonais/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/ultraestrutura , Clonagem Molecular , Microscopia Crioeletrônica , Cristalografia por Raios X , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/farmacologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/farmacologia , Região Variável de Imunoglobulina/ultraestrutura , Simulação de Dinâmica Molecular , Oócitos , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/ultraestrutura , Células Sf9 , Spodoptera , Xenopus laevis
4.
Methods Enzymol ; 653: 3-19, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34099177

RESUMO

Despite major advances in methodologies for membrane protein production over the last two decades, there remain challenging protein complexes that are technically difficult to yield by conventional recombinant expression methods. A large number of these proteins are multimeric membrane proteins from eukaryotic species, which are required to pass through stringent quality control mechanisms of host cells for proper folding and complex assembly. Here, we describe the development procedure to improve the production efficiency of multi-oligomeric membrane protein complexes in insect cells and recombinant baculovirus, which involves screening of promoters, enhancers, and untranslated regions for expression levels, using calcium homeostasis modulator (CALHM) and N-methyl-d-aspartate receptor (NMDAR) proteins as examples. We demonstrate that our insect cell expression strategy is effective in expression of both multi-homomeric CALHM proteins and multi-heteromeric NMDARs.


Assuntos
Baculoviridae , Proteínas de Membrana , Animais , Baculoviridae/genética , Insetos , Proteínas de Membrana/genética , Receptores de N-Metil-D-Aspartato
5.
Nat Struct Mol Biol ; 27(3): 305, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32066965

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

6.
Nat Struct Mol Biol ; 27(2): 150-159, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31988524

RESUMO

The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), through which ions and ATP permeate in a voltage-dependent manner to control neuronal excitability, taste signaling and pathologies of depression and Alzheimer's disease. Despite such critical biological roles, the structures and patterns of their oligomeric assembly remain unclear. Here, we reveal the structures of two CALHMs, chicken CALHM1 and human CALHM2, by single-particle cryo-electron microscopy (cryo-EM), which show novel assembly of the four transmembrane helices into channels of octamers and undecamers, respectively. Furthermore, molecular dynamics simulations suggest that lipids can favorably assemble into a bilayer within the larger CALHM2 pore, but not within CALHM1, demonstrating the potential correlation between pore size, lipid accommodation and channel activity.


Assuntos
Proteínas Aviárias/metabolismo , Canais de Cálcio/metabolismo , Galinhas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/química , Cálcio/metabolismo , Canais de Cálcio/química , Microscopia Crioeletrônica , Homeostase , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
7.
Nat Commun ; 11(1): 423, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969570

RESUMO

N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the L-glutamate binding site can be targeted for GluN2C/2D-specific inhibition.


Assuntos
Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Animais , Sítios de Ligação , Ligação Competitiva , Ácido Glutâmico/química , Ácido Glutâmico/metabolismo , Cinética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Xenopus laevis
8.
Neuron ; 92(6): 1324-1336, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27916457

RESUMO

Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino-terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete because of the lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc-binding site and reveal distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability, supporting the general model in which the bi-lobe motion in ATD regulates the channel activity in NMDA receptors.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Zinco/metabolismo , 2-Hidroxifenetilamina/metabolismo , Animais , Sítios de Ligação , Western Blotting , Cristalografia , Ligação de Hidrogênio , Piperidinas/farmacologia , Estrutura Quaternária de Proteína , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Células Sf9 , Spodoptera , Xenopus laevis , Zinco/farmacologia
9.
Nature ; 534(7605): 63-8, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27135925

RESUMO

The physiology of N-methyl-d-aspartate (NMDA) receptors is fundamental to brain development and function. NMDA receptors are ionotropic glutamate receptors that function as heterotetramers composed mainly of GluN1 and GluN2 subunits. Activation of NMDA receptors requires binding of neurotransmitter agonists to a ligand-binding domain (LBD) and structural rearrangement of an amino-terminal domain (ATD). Recent crystal structures of GluN1-GluN2B NMDA receptors bound to agonists and an allosteric inhibitor, ifenprodil, represent the allosterically inhibited state. However, how the ATD and LBD move to activate the NMDA receptor ion channel remains unclear. Here we applied X-ray crystallography, single-particle electron cryomicroscopy and electrophysiology to rat NMDA receptors to show that, in the absence of ifenprodil, the bi-lobed structure of GluN2 ATD adopts an open conformation accompanied by rearrangement of the GluN1-GluN2 ATD heterodimeric interface, altering subunit orientation in the ATD and LBD and forming an active receptor conformation that gates the ion channel.


Assuntos
Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , Eletrofisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestrutura
10.
Nature ; 475(7355): 249-53, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21677647

RESUMO

Since it was discovered that the anti-hypertensive agent ifenprodil has neuroprotective activity through its effects on NMDA (N-methyl-D-aspartate) receptors, a determined effort has been made to understand the mechanism of action and to develop improved therapeutic compounds on the basis of this knowledge. Neurotransmission mediated by NMDA receptors is essential for basic brain development and function. These receptors form heteromeric ion channels and become activated after concurrent binding of glycine and glutamate to the GluN1 and GluN2 subunits, respectively. A functional hallmark of NMDA receptors is that their ion-channel activity is allosterically regulated by binding of small compounds to the amino-terminal domain (ATD) in a subtype-specific manner. Ifenprodil and related phenylethanolamine compounds, which specifically inhibit GluN1 and GluN2B NMDA receptors, have been intensely studied for their potential use in the treatment of various neurological disorders and diseases, including depression, Alzheimer's disease and Parkinson's disease. Despite considerable enthusiasm, mechanisms underlying the recognition of phenylethanolamines and ATD-mediated allosteric inhibition remain limited owing to a lack of structural information. Here we report that the GluN1 and GluN2B ATDs form a heterodimer and that phenylethanolamine binds at the interface between GluN1 and GluN2B, rather than within the GluN2B cleft. The crystal structure of the heterodimer formed between the GluN1b ATD from Xenopus laevis and the GluN2B ATD from Rattus norvegicus shows a highly distinct pattern of subunit arrangement that is different from the arrangements observed in homodimeric non-NMDA receptors and reveals the molecular determinants for phenylethanolamine binding. Restriction of domain movement in the bi-lobed structure of the GluN2B ATD, by engineering of an inter-subunit disulphide bond, markedly decreases sensitivity to ifenprodil, indicating that conformational freedom in the GluN2B ATD is essential for ifenprodil-mediated allosteric inhibition of NMDA receptors. These findings pave the way for improving the design of subtype-specific compounds with therapeutic value for neurological disorders and diseases.


Assuntos
2-Hidroxifenetilamina/metabolismo , Piperidinas/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , 2-Hidroxifenetilamina/química , 2-Hidroxifenetilamina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Movimento , Fármacos Neuroprotetores/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Multimerização Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Xenopus laevis
11.
Nat Commun ; 2: 294, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21522138

RESUMO

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors that mediate a majority of excitatory synaptic transmission. One unique property of GluN1/GluN2D NMDA receptors is an unusually prolonged deactivation time course following the removal of L-glutamate. Here we show, using x-ray crystallography and electrophysiology, that the deactivation time course of GluN1/GluN2D receptors is influenced by the conformational variability of the ligand-binding domain (LBD) as well as the structure of the activating ligand. L-glutamate and L-CCG-IV induce significantly slower deactivation time courses compared with other agonists. Crystal structures of the isolated GluN2D LBD in complex with various ligands reveal that the binding of L-glutamate induces a unique conformation at the backside of the ligand-binding site in proximity to the region at which the transmembrane domain would be located in the intact receptors. These data suggest that the activity of the GluN1/GluN2D NMDA receptor is controlled distinctively by the endogenous neurotransmitter L-glutamate.


Assuntos
Ácido Glutâmico/química , Receptores de N-Metil-D-Aspartato/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Conformação Molecular , Dados de Sequência Molecular , Conformação Proteica , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
EMBO J ; 28(24): 3910-20, 2009 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-19910922

RESUMO

N-methyl-D-aspartate (NMDA) receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of fast excitatory synaptic transmission in the mammalian brain. One of the hallmarks for the function of NMDA receptors is that their ion channel activity is allosterically regulated by binding of modulator compounds to the extracellular amino-terminal domain (ATD) distinct from the L-glutamate-binding domain. The molecular basis for the ATD-mediated allosteric regulation has been enigmatic because of a complete lack of structural information on NMDA receptor ATDs. Here, we report the crystal structures of ATD from the NR2B NMDA receptor subunit in the zinc-free and zinc-bound states. The structures reveal the overall clamshell-like architecture distinct from the non-NMDA receptor ATDs and molecular determinants for the zinc-binding site, ion-binding sites, and the architecture of the putative phenylethanolamine-binding site.


Assuntos
Receptores de N-Metil-D-Aspartato/química , Sítio Alostérico , Animais , Encéfalo/embriologia , Cristalografia por Raios X/métodos , Eletrofisiologia/métodos , Feminino , Ácido Glutâmico/química , Conformação Molecular , Oócitos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Xenopus laevis/metabolismo , Zinco/química
13.
J Mol Biol ; 364(3): 302-8, 2006 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-17023000

RESUMO

DBC2 is a tumor suppressor gene linked to breast and lung cancers. Although DBC2 belongs to the RHO GTPase family, it has a unique structure that contains a Broad-Complex/Tramtrack/Bric a Brac (BTB) domain at the C terminus instead of a typical CAAX motif. A limited number of functional studies on DBC2 have indicated its participation in diverse cellular activities, such as ubiquitination, cell-cycle control, cytoskeleton organization and protein transport. In this study, the role of DBC2 in protein transport was analyzed using vesicular stomatitis virus glycoprotein (VSVG) fused with green fluorescent protein. We discovered that DBC2 knockdown hinders the VSVG transport system in 293 cells. Previous studies have demonstrated that VSVG is transported via the microtubule motor complex. We demonstrate that DBC2 mobility depends also on an intact microtubule network. We conclude that DBC2 plays an essential role in microtubule-mediated VSVG transport from the endoplasmic reticulum to the Golgi apparatus.


Assuntos
Proteínas de Ligação ao GTP/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Proteínas do Envelope Viral/fisiologia , Motivos de Aminoácidos , Linhagem Celular , Retículo Endoplasmático/metabolismo , Proteínas de Ligação ao GTP/genética , Complexo de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Microtúbulos/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Supressoras de Tumor/genética
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