RESUMEN
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
Asunto(s)
Receptores de Glutamato , Receptores Ionotrópicos de Glutamato , Animales , Sistema Nervioso Central , Ácido Glutámico , Humanos , Neurotransmisores , Receptores Ionotrópicos de Glutamato/genéticaRESUMEN
The biological phenomenon of protein-lipid interactions in cell membranes underlies the diversity of peripheral membrane protein function and physical properties of the membrane. To summarize novel findings in the field, this research highlight focuses on recent publications in Biophysical Journal.
RESUMEN
Kainate receptors are a subtype of ionotropic glutamate receptors that form transmembrane channels upon binding glutamate. Here, we have investigated the mechanism of partial agonism in heteromeric GluK2/K5 receptors, where the GluK2 and GluK5 subunits have distinct agonist binding profiles. Using single-molecule Förster resonance energy transfer, we found that at the bi-lobed agonist-binding domain, the partial agonist AMPA-bound receptor occupied intermediate cleft closure conformational states at the GluK2 cleft, compared to the more open cleft conformations in apo form and more closed cleft conformations in the full agonist glutamate-bound form. In contrast, there is no significant difference in cleft closure states at the GluK5 agonist-binding domain between the partial agonist AMPA- and full agonist glutamate-bound states. Additionally, unlike the glutamate-bound state, the dimer interface at the agonist-binding domain is not decoupled in the AMPA-bound state. Our findings suggest that partial agonism observed with AMPA binding is mediated primarily due to differences in the GluK2 subunit, highlighting the distinct contributions of the subunits towards activation.
RESUMEN
Allostery can be manifested as a combination of repression and activation in multidomain proteins allowing for fine tuning of regulatory mechanisms. Here we have used single molecule fluorescence resonance energy transfer (smFRET) and molecular dynamics simulations to study the mechanism of allostery underlying negative cooperativity between the two agonists glutamate and glycine in the NMDA receptor. These data show that binding of one agonist leads to conformational flexibility and an increase in conformational spread at the second agonist site. Mutational and cross-linking studies show that the dimer-dimer interface at the agonist-binding domain mediates the allostery underlying the negative cooperativity. smFRET on the transmembrane segments shows that they are tightly coupled in the unliganded and single agonist-bound form and only upon binding both agonists the transmembrane domain explores looser packing which would facilitate activation.
Asunto(s)
Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Dimerización , Ácido Glutámico/química , Ácido Glutámico/metabolismo , Glicina/química , Glicina/metabolismo , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Dominios Proteicos , Ratas , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
Zn2+ has been shown to have a wide range of modulatory effects on neuronal AMPARs. However, the mechanism of modulation is largely unknown. Here we show that Zn2+ inhibits GluA2(Q) homomeric receptors in an activity- and voltage-dependent manner, indicating a pore block mechanism. The rate of inhibition is slow, in the hundreds of milliseconds at millimolar Zn2+ concentrations; hence, the inhibition is only observed in the residual nondesensitizing currents. Consequently, the inhibition is higher for GluA2 receptors in complex with auxiliary subunits γ2 and γ8 where the residual activation is larger. The extent of inhibition is also dependent on charge at site 607, the site that undergoes RNA editing in GluA2 subunits replacing glutamine to arginine, with the percent inhibition being lower and IC50 being higher for the edited GluA2(R) relative to unedited GluA2(Q) and to GluA2(Q607E), a mutation observed in the genetic screen of a patient exhibiting developmental delays. We also show that Zn2+ inhibition is significant during rapid repetitive activity with pulses of millimolar concentrations of glutamate in both receptors expressed in HEK cells as well as in native receptors in cortical neurons of C57BL/6J mice of either sex, indicating a physiological relevance of this inhibition.SIGNIFICANCE STATEMENT Zn2+ is present along with glutamate in synaptic vesicles and coreleased during synaptic transmission, modulating the postsynaptic ionotropic glutamate receptors. While Zn2+ inhibition of the NMDA subtype of the ionotropic glutamate receptors is well characterized, the mechanism of modulation of the AMPA subtype is much less known. Here we have systematically studied Zn2+ inhibition of AMPARs by varying calcium permeability, auxiliary subunits, and activation levels and show that Zn2+ inhibits AMPARs in an activity-dependent manner, opening up this pathway as a means to pharmacologically modulate the receptors.
Asunto(s)
Receptores AMPA/antagonistas & inhibidores , Zinc/farmacología , Animales , Corteza Cerebral/metabolismo , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Edición de ARN , Receptores AMPA/genética , TransfecciónRESUMEN
N-methyl-D-aspartate (NMDA) receptors mediate synaptic excitatory signaling in the mammalian central nervous system by forming calcium-permeable transmembrane channels upon binding glutamate and coagonist glycine. Ca2+ influx through NMDA receptors leads to channel inactivation through a process mediated by resident calmodulin bound to the intracellular C-terminal segment of the GluN1 subunit of the receptor. Using single-molecule FRET investigations, we show that in the presence of calcium-calmodulin, the distance across the two GluN1 subunits at the entrance of the first transmembrane segment is shorter and the bilobed cleft of the glycine-binding domain in GluN1 is more closed when bound to glycine and glutamate relative to what is observed in the presence of barium-calmodulin. Consistent with these observations, the glycine deactivation rate is slower in the presence of calcium-calmodulin. Taken together, these results show that the binding of calcium-calmodulin to the C-terminus has long-range allosteric effects on the extracellular segments of the receptor that may contribute to the calcium-dependent inactivation.
Asunto(s)
Calcio , Receptores de N-Metil-D-Aspartato , Animales , Calcio/metabolismo , Calmodulina , Ácido Glutámico , GlicinaRESUMEN
Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas , Microscopía por Crioelectrón , Glutamatos , Conformación MolecularRESUMEN
Acid-sensing ion channels (ASICs) are trimeric cation-selective ion channels activated by protons in the physiological range. Recent reports have revealed that postsynaptically localized ASICs contribute to the excitatory postsynaptic current by responding to the transient acidification of the synaptic cleft that accompanies neurotransmission. In response to such brief acidic transients, both recombinant and native ASICs show extremely rapid deactivation in outside-out patches when jumping from a pH 5 stimulus to a single resting pH of 8. Given that the resting pH of the synaptic cleft is highly dynamic and depends on recent synaptic activity, we explored the kinetics of ASIC1a and 1a/2a heteromers to such brief pH transients over a wider [H+] range to approximate neuronal conditions better. Surprisingly, the deactivation of ASICs was steeply dependent on the pH, spanning nearly three orders of magnitude from extremely fast (<1 ms) at pH 8 to very slow (>300 ms) at pH 7. This study provides an example of a ligand-gated ion channel whose deactivation is sensitive to agonist concentrations that do not directly activate the receptor. Kinetic simulations and further mutagenesis provide evidence that ASICs show such steeply agonist-dependent deactivation because of strong cooperativity in proton binding. This capacity to signal across such a large synaptically relevant bandwidth enhances the response to small-amplitude acidifications likely to occur at the cleft and may provide ASICs with the ability to shape activity in response to the recent history of the synapse.
Asunto(s)
Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/química , Canales Iónicos Sensibles al Ácido/genética , Animales , Células CHO , Pollos , Cricetinae , Cricetulus , Humanos , Concentración de Iones de Hidrógeno , Cinética , Canales Iónicos Activados por Ligandos/metabolismo , Protones , Ratas , Sinapsis/metabolismo , Transmisión SinápticaRESUMEN
Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal ß-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 µs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.
Asunto(s)
Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas p21(ras)/química , Animales , Línea Celular , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia , Mutación Missense , Nanoestructuras/química , Fosfatidilcolinas/química , Fosfatidilserinas/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Imagen Individual de MoléculaRESUMEN
N-Methyl-D-aspartate (NMDA) receptors are the main calcium-permeable excitatory receptors in the mammalian central nervous system. The NMDA receptor gating is complex, exhibiting multiple closed, open, and desensitized states; however, central questions regarding the conformations and energetics of the transmembrane domains as they relate to the gating states are still unanswered. Here, using single-molecule Förster resonance energy transfer (smFRET), we map the energy landscape of the first transmembrane segment of the Rattus norvegicus NMDA receptor under resting and various liganded conditions. These results show kinetically and structurally distinct changes associated with apo, agonist-bound, and inhibited receptors linked by a linear mechanism of gating at this site. Furthermore, the smFRET data suggest that allosteric inhibition by zinc occurs by an uncoupling of the agonist-induced changes at the extracellular domains from the gating motions leading to an apo-like state, while dizocilpine, a pore blocker, stabilizes multiple closely packed transmembrane states.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Maleato de Dizocilpina/farmacología , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Zinc/farmacologíaRESUMEN
Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.
Asunto(s)
Membrana Celular/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Proteínas de la Membrana/química , Procesamiento Proteico-Postraduccional , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Fosforilación , Dominios ProteicosRESUMEN
A fundamental objective in membrane biology is to understand and predict how a protein sequence folds and orients in a lipid bilayer. Establishing the principles governing membrane protein folding is central to understanding the molecular basis for membrane proteins that display multiple topologies, the intrinsic dynamic organization of membrane proteins, and membrane protein conformational disorders resulting in disease. We previously established that lactose permease of Escherichia coli displays a mixture of topological conformations and undergoes postassembly bidirectional changes in orientation within the lipid bilayer triggered by a change in membrane phosphatidylethanolamine content, both in vivo and in vitro. However, the physiological implications and mechanism of dynamic structural reorganization of membrane proteins due to changes in lipid environment are limited by the lack of approaches addressing the kinetic parameters of transmembrane protein flipping. In this study, real-time fluorescence spectroscopy was used to determine the rates of protein flipping in the lipid bilayer in both directions and transbilayer flipping of lipids triggered by a change in proteoliposome lipid composition. Our results provide, for the first time to our knowledge, a dynamic picture of these events and demonstrate that membrane protein topological rearrangements in response to lipid modulations occur rapidly following a threshold change in proteoliposome lipid composition. Protein flipping was not accompanied by extensive lipid-dependent unfolding of transmembrane domains. Establishment of lipid bilayer asymmetry was not required but may accelerate the rate of protein flipping. Membrane protein flipping was found to accelerate the rate of transbilayer flipping of lipids.
Asunto(s)
Proteínas de la Membrana/química , Fosfolípidos/química , Escherichia coli/enzimología , Transferencia Resonante de Energía de Fluorescencia , Membrana Dobles de Lípidos , Proteínas de Transporte de Membrana/química , Proteolípidos , Espectrometría de FluorescenciaRESUMEN
The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Receptores de N-Metil-D-Aspartato/química , Animales , Línea Celular , Humanos , Dominios Proteicos , RatasRESUMEN
KEY POINTS: Acid-sensing ion channels (ASICs) act as neurotransmitter receptors by responding to synaptic cleft acidification. We investigated how ASIC1a homomers and ASIC1a/2a heteromers respond to brief stimuli, jumping from pH 8.0 to 5.0, approximating the time course of neurotransmitter in the cleft. We find that ASICs deactivate surprisingly fast in response to such brief stimuli from pH 8.0 to 5.0, whereas they desensitize comparatively slowly to prolonged activation. The combination of unusually fast deactivation with slow desensitzation enables recombinant ASIC1a homomers and ASIC1a/2a heteromers, as well as native ASICs of sensory neurons, to follow trains of such brief pH 8.0 to 5.0 stimuli at high frequencies. This capacity for high-frequency signalling persists under a physiological pH of 7.4 with ASIC1a/2a heteromers, suggesting that they may sustain postsynaptic responses when other receptors desensitize. ABSTRACT: The neurotransmitter-gated ion channels that underlie rapid synaptic transmission are often subjected to bursts of very brief neurotransmitter release at high frequencies. When challenged with such short duration high-frequency stimuli, neurotransmitter-gated ion channels generally exhibit the common response of desensitization. Recently, acid-sensing ion channels (ASICs) were shown to act as neurotransmitter-gated ion channels because postsynaptic ASICs can be activated by the transient acidification of the synaptic cleft accompanying neurotransmission. In the present study, we examined the responses of recombinant ASIC1a homomers, ASIC1a/2a heteromers and native ASICs from sensory neurons to 1 ms acidification stimuli, switching from pH 8.0 to 5.0, as either single pulses or trains of pulses at physiologically relevant frequencies. We found that ASIC deactivation is extremely fast and, in contrast to most other neurotransmitter-gated ion channels, ASICs show no desensitization during high-frequency stimulus trains under these conditions. We also found that accelerating ASIC desensitization by anion substitution can induce depression during high-frequency trains. When using a baseline physiological pH of 7.4, the ASIC1a responses were too small to reliably measure, presumably as a result of steady-state desensitization. However, ASIC1a/2 heteromers gave robust responses when using a baseline pH of 7.4 and were also able to sustain these responses during high-frequency stimulus trains. In conclusion, we report that the slow desensitization and fast deactivation of ASIC1a/2a heteromers enables them to sustain postsynaptic responses to bursts at high frequencies at a physiological pH that may desensitize other receptors.
Asunto(s)
Canales Iónicos Sensibles al Ácido/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Ganglios Espinales/fisiología , Ratones , Ratones Endogámicos C57BL , Ratas , Transmisión Sináptica/fisiologíaRESUMEN
The N-methyl-d-aspartate (NMDA) subtype of the ionotropic glutamate receptors is the primary mediator of calcium-permeable excitatory neurotransmission in the central nervous system. Subunit composition and binding of allosteric modulators to the amino-terminal domain determine the open probability of the channel. By using luminescence resonance energy transfer with functional receptors expressed in CHO cells, we show that the cleft of the amino-terminal domain of the GluN2B subunit, which has a lower channel open probability, is on average more closed than the GluN2A subunit, which has a higher open probability. Furthermore, the GluN1 amino-terminal domain adopts a more open conformation when coassembled with GluN2A than with GluN2B. Binding of spermine, an allosteric potentiator, opens the amino-terminal domain cleft of both the GluN2B subunit and the adjacent GluN1 subunit. These studies provide direct structural evidence that the inherent conformations of the amino-terminal domains vary based on the subunit and match the reported open probabilities for the receptor.
Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Espermina/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Células CHO , Cricetinae , Cricetulus , Activación del Canal Iónico/genética , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and L-alanine, and full agonists glycine and D-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-L-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation.
Asunto(s)
Glicina/química , Conformación Proteica , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Alanina/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Glicina/metabolismo , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Receptores de N-Metil-D-Aspartato/agonistas , Relación Estructura-ActividadRESUMEN
The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.