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The Journal of Neuroscience : the... Apr 2011Earlier findings had suggested that spontaneous and evoked glutamate release activates non-overlapping populations of NMDA receptors. Here, we evaluated whether AMPA...
Earlier findings had suggested that spontaneous and evoked glutamate release activates non-overlapping populations of NMDA receptors. Here, we evaluated whether AMPA receptor populations activated by spontaneous and evoked release show a similar segregation. To track the receptors involved in spontaneous or evoked neurotransmission, we used a polyamine agent, philanthotoxin, that selectively blocks AMPA receptors lacking GluR2 subunits in a use-dependent manner. In hippocampal neurons obtained from GluR2-deficient mice, philanthotoxin application decreased AMPA-receptor-mediated spontaneous miniature EPSCs (AMPA-mEPSCs) down to 20% of their initial level within 5 min. In contrast, the same philanthotoxin application at rest decreased the subsequent AMPA-receptor-mediated evoked EPSCs (eEPSCs) only down to 80% of their initial value. A 10-min-long perfusion of philanthotoxin further decreased AMPA-eEPSC amplitudes to 60% of their initial magnitude, which remained substantially higher than the level of AMPA-mEPSC block achieved within 5 min. Finally, stimulation after removal of philanthotoxin resulted in reversal of AMPA-eEPSC block, verifying strict use dependence of philanthotoxin. These results support the notion that spontaneous and evoked neurotransmission activate distinct sets of AMPA receptors and bolster the hypothesis that synapses harbor separate microdomains of evoked and spontaneous signaling.
Topics: Anesthetics, Local; Animals; Animals, Newborn; Cells, Cultured; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Glutamic Acid; Hippocampus; Lidocaine; Male; Mice; Mice, Knockout; Neurons; Patch-Clamp Techniques; Polyamines; Receptors, AMPA; Tetrodotoxin; Valine
PubMed: 21471372
DOI: 10.1523/JNEUROSCI.5234-10.2011 -
Annual Review of Physiology 2013X-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... (Review)
Review
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.
Topics: Animals; Computer Simulation; Crystallography, X-Ray; Humans; Ion Channels; Receptors, AMPA; Receptors, Glutamate; Receptors, Ionotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Signal Transduction
PubMed: 22974439
DOI: 10.1146/annurev-physiol-030212-183711 -
Future Medicinal Chemistry Mar 2019AMPA receptor antagonists are drug candidates for potential treatment of a number of CNS diseases that involve excessive receptor activation. To date, small-molecule... (Review)
Review
AMPA receptor antagonists are drug candidates for potential treatment of a number of CNS diseases that involve excessive receptor activation. To date, small-molecule compounds are the dominating drug candidates in the field. However, lower potency, cross activity and poor water solubility are generally associated with these compounds. Here we show the potential of RNA-based antagonists or RNA aptamers as drug candidates and some strategies to discover these aptamers from a random sequence library (∼10 sequences). As an alternative to small molecule compounds, our aptamers exhibit higher potency and selectivity toward AMPA receptors. Because aptamers are RNA molecules, they are naturally water soluble. We also discuss the major challenges of translating RNA aptamers as lead molecules into drugs/treatment options.
Topics: Animals; Aptamers, Nucleotide; Drug Development; Drug Discovery; Humans; Nervous System Diseases; Nucleic Acid Conformation; Receptors, AMPA; SELEX Aptamer Technique
PubMed: 30912676
DOI: 10.4155/fmc-2018-0364 -
British Journal of Pharmacology Jul 2022AMPA receptors, which shape excitatory postsynaptic currents and are directly involved in overactivation of synaptic function during seizures, represent a well-accepted...
BACKGROUND AND PURPOSE
AMPA receptors, which shape excitatory postsynaptic currents and are directly involved in overactivation of synaptic function during seizures, represent a well-accepted target for anti-epileptic drugs. Trans-4-butylcyclohexane carboxylic acid (4-BCCA) has emerged as a new promising anti-epileptic drug in several in vitro and in vivo seizure models, but the mechanism of its action remained unknown. The purpose of this study is to characterize structure and dynamics of 4-BCCA interaction with AMPA receptors.
EXPERIMENTAL APPROACH
We studied the molecular mechanism of AMPA receptor inhibition by 4-BCCA using a combination of X-ray crystallography, mutagenesis, electrophysiological assays, and molecular dynamics simulations.
KEY RESULTS
We identified 4-BCCA binding sites in the transmembrane domain (TMD) of AMPA receptor, at the lateral portals formed by transmembrane segments M1-M4. At this binding site, 4-BCCA is very dynamic, assumes multiple poses, and can enter the ion channel pore.
CONCLUSION AND IMPLICATIONS
4-BCCA represents a low-affinity inhibitor of AMPA receptors that acts at the TMD sites distinct from non-competitive inhibitors, such as the anti-epileptic drug perampanel and the ion channel blockers. Further studies might examine the possibsility of synergistic use of these inhibitors in treatment of epilepsy and a wide range of neurological disorders and gliomas.
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.
Topics: Carboxylic Acids; Cyclohexanes; Humans; Receptors, AMPA; Seizures
PubMed: 32959886
DOI: 10.1111/bph.15254 -
Biophysical Journal Jul 2020Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Desensitization of the...
Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the central nervous system. Desensitization of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype after glutamate binding appears critical for brain function and involves rearrangement of the ligand binding domains (LBDs). Recently, several full-length structures of ionotropic glutamate receptors in putative desensitized states were published. These structures indicate movements of the LBDs that might be trapped by cysteine cross-links and metal bridges. We found that cysteine mutants at the interface between subunits A and C and lateral zinc bridges (between subunits C and D or A and B) can trap freely desensitizing receptors in a spectrum of states with different stabilities. Consistent with a close approach of subunits during desensitization processes, the introduction of bulky amino acids at the A-C interface produced a receptor with slow recovery from desensitization. Further, in wild-type GluA2 receptors, we detected the population of a stable desensitized state with a lifetime around 1 s. Using mutations that progressively stabilize deep desensitized states (E713T and Y768R), we were able to selectively protect receptors from cross-links at both the diagonal and lateral interfaces. Ultrafast perfusion enabled us to perform chemical modification in less than 10 ms, reporting movements associated to desensitization on this timescale within LBD dimers in resting receptors. These observations suggest that small disruptions of quaternary structure are sufficient for fast desensitization and that substantial rearrangements likely correspond to stable desensitized states that are adopted relatively slowly on a timescale much longer than physiological receptor activation.
Topics: Mutation; Protein Domains; Receptors, AMPA; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
PubMed: 32559412
DOI: 10.1016/j.bpj.2020.05.029 -
PloS One 2012Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory...
Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.
Topics: Animals; Calcium Channels; Dose-Response Relationship, Drug; Glutamic Acid; HEK293 Cells; Humans; Immunoblotting; Immunoprecipitation; Ion Channel Gating; Multiprotein Complexes; Patch-Clamp Techniques; Protein Conformation; Rats; Receptors, AMPA
PubMed: 23166629
DOI: 10.1371/journal.pone.0049282 -
Cell Aug 2014Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to...
Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.
Topics: Animals; Cryoelectron Microscopy; Crystallography, X-Ray; Fluorouracil; Gene Knockout Techniques; Kainic Acid; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Tertiary; Rats; Receptors, AMPA
PubMed: 25109876
DOI: 10.1016/j.cell.2014.07.023 -
Molecular Pain Jan 2010The functional properties of alpha-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptors in different brain regions, such as hippocampus and cerebellum, have... (Review)
Review
The functional properties of alpha-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptors in different brain regions, such as hippocampus and cerebellum, have been well studied in vitro and in vivo. The AMPA receptors present a unique characteristic in the mechanisms of subunit regulation during LTP (long-term potentiation) and LTD (long-term depression), which are involved in the trafficking, altered composition and phosphorylation of AMPA receptor subunits. Accumulated data have demonstrated that spinal AMPA receptors play a critical role in the mechanism of both acute and persistent pain. However, less is known about the biochemical regulation of AMPA receptor subunits in the spinal cord in response to painful stimuli. Recent studies have shown that some important regulatory processes, such as the trafficking of AMPA receptor subunit, subunit compositional changes, phosphorylation of AMPA receptor subunits, and their interaction with partner proteins may contribute to spinal nociceptive transmission. Of all these regulation processes, the phosphorylation of AMPA receptor subunits is the most important since it may trigger or affect other cellular processes. Therefore, these study results may suggest an effective strategy in developing novel analgesics targeting AMPA receptor subunit regulation that may be useful in treating persistent and chronic pain without unacceptable side effects in the clinics.
Topics: Animals; Glutamic Acid; Humans; Pain; Phosphorylation; Protein Subunits; Protein Transport; Receptors, AMPA; Sensory Receptor Cells; Spinal Cord; Synaptic Transmission
PubMed: 20092646
DOI: 10.1186/1744-8069-6-5 -
Neuron Oct 2003Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate... (Review)
Review
Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.
Topics: Amino Acid Sequence; Animals; Binding Sites; Excitatory Postsynaptic Potentials; Humans; Molecular Sequence Data; Protein Transport; Receptors, AMPA; Synapses
PubMed: 14556714
DOI: 10.1016/s0896-6273(03)00640-8 -
Cell Reports Oct 2016Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary...
Fast excitatory synaptic signaling in the mammalian brain is mediated by AMPA-type ionotropic glutamate receptors. In neurons, AMPA receptors co-assemble with auxiliary proteins, such as stargazin, which can markedly alter receptor trafficking and gating. Here, we used luminescence resonance energy transfer measurements to map distances between the full-length, functional AMPA receptor and stargazin expressed in HEK293 cells and to determine the ensemble structural changes in the receptor due to stargazin. In addition, we used single-molecule fluorescence resonance energy transfer to study the structural and conformational distribution of the receptor and how this distribution is affected by stargazin. Our nanopositioning data place stargazin below the AMPA receptor ligand-binding domain, where it is well poised to act as a scaffold to facilitate the long-range conformational selection observations seen in single-molecule experiments. These data support a model of stargazin acting to stabilize or select conformational states that favor activation.
Topics: Calcium Channels; HEK293 Cells; Humans; Ligands; Neurons; Protein Binding; Protein Domains; Protein Transport; Receptors, AMPA; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
PubMed: 27705782
DOI: 10.1016/j.celrep.2016.09.014