Noncompetitive antagonists induce cooperative AMPA receptor channel gating

Glutamate is released from presynaptic nerve terminals in the central nervous system (CNS) and spreads excitation by binding to and activating postsynaptic iGluRs. Of the potential glutamate targets, tetrameric AMPA receptors mediate fast, transient CNS signaling. Each of the four AMPA subunits in the receptor channel complex is capable of binding glutamate at its ligand-binding domains and transmitting the energy of activation to the pore domain. Homotetrameric AMPA receptor channels open in a stepwise manner, consistent with independent activation of individual subunits, and they exhibit complex kinetic behavior that manifests as temporal shifts between four different conductance levels. Here, we investigate how two AMPA receptor-selective noncompetitive antagonists, GYKI-52466 and GYKI-53655, disrupt the intrinsic step-like gating patterns of maximally activated homotetrameric GluA3 receptors using single-channel recordings from cell-attached patches. Interactions of these 2,3-benzodiazepines with residues in the boundary between the extracellular linkers and transmembrane helical domains reorganize the gating behavior of channels. Low concentrations of modulators stabilize open and closed states to different degrees and coordinate the activation of subunits so that channels open directly from closed to higher conductance levels. Using kinetic and structural models, we provide insight into how the altered gating patterns might arise from molecular contacts within the extracellular linker-channel boundary. Our results suggest that this region may be a tunable locus for AMPA receptor channel gating.

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The action of Con-ikot-ikot toxin on single AMPA-type glutamate receptors

10.1101/2021.02.19.432041 ◽

2021 ◽

Author(s):

Jelena Baranovic ◽

Sebastian Braunbeck ◽

Nikolai Zaki ◽

Sonja Minniberger ◽

Miriam Chebli ◽

...

Keyword(s):

Ligand Binding ◽

Glutamate Receptors ◽

Ampa Receptor ◽

Ampa Receptors ◽

Single Channel ◽

Basic Research ◽

High Specificity ◽

Direct Consequence ◽

Binding Mode ◽

Binding Domains

SummaryConotoxins are a large group of naturally occurring toxic peptides produced by the predatory sea snails of the genus Conus. Many of these toxins target ion channels, often with high specificity and affinity. As such, they have proven to be invaluable for basic research as well as acting as leads for therapeutic strategies. Con-ikot-ikot is the only conotoxin so far identified that targets AMPA-type glutamate receptors, the main mediators of excitatory neurotransmission in the vertebrate brain. Here, we describe how the toxin modifies the activity of AMPA receptors at the single-channel level. The toxin binds to the AMPA receptor with high affinity (EC50 = 5 nM) and once bound, takes minutes to wash out. As shown previously, it effectively blocks desensitization of AMPA receptors, however, compared to other desensitisation blockers, it is a poor stabiliser of the open channel because toxin-bound AMPA receptors undergo frequent, brief closures. We propose this is a direct consequence of its unique binding mode to the ligand binding domains. Unlike other blockers of desensitization, which stabilise individual dimers within an AMPA receptor tetramer, the toxin immobilizes all four ligand binding domains of the tetramer. This result further emphasises that quaternary reorganization of independent LBD dimers is essential for the full activity of AMPA receptors.

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Gadolinium Reduces AMPA Receptor Desensitization and Deactivation in Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.2001.86.1.173 ◽

2001 ◽

Vol 86 (1) ◽

pp. 173-182 ◽

Cited By ~ 3

Author(s):

Saobo Lei ◽

John F. MacDonald

Keyword(s):

Steady State ◽

Ampa Receptor ◽

Hippocampal Neurons ◽

Time Course ◽

Single Channel ◽

Trivalent Cation ◽

Receptor Desensitization ◽

Whole Cell ◽

Low Concentrations ◽

Cultured Hippocampal Neurons

The actions of the trivalent cation Gd3+ on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd3+ (0.1–10 μM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 μM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd3+also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd3+ reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd3+ inhibited both peak and steady-state currents, indicating that Gd3+ both reduces desensitization and inhibits these currents. Gd3+ reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd3+. The Gd3+-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd3+ reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd3+ potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.

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Comparison of the Effects of Convulsant and Depressant Barbiturate Stereoisomers on AMPA-type Glutamate Receptors

Anesthesiology ◽

10.1097/00000542-199906000-00028 ◽

1999 ◽

Vol 90 (6) ◽

pp. 1704-1713. ◽

Cited By ~ 23

Author(s):

Yoshinori Kamiya ◽

Tomio Andoh ◽

Ryosuke Furuya ◽

Satoshi Hattori ◽

Itaru Watanabe ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Glutamate Receptors ◽

Ampa Receptor ◽

Ampa Receptors ◽

Dependent Manner ◽

Induced Current ◽

Gaba A ◽

The Central Nervous System

Background Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Although barbiturates have been shown to suppress the AMPA receptor-mediated responses, it is unclear whether this effect contributes to the anesthetic action of barbiturates. The authors compared the effects of depressant [R(-)] and convulsant [S(+)] stereoisomers of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) on the AMPA and gamma-aminobutyric acid type A (GABA(A)) receptor-mediated currents to determine if the inhibitory effects on AMPA receptors correlate to the in vivo effects of the isomers. Method The authors measured whole-cell currents in the rat cultured cortical neuron at holding potential of -60 mV. Kainate 500 microM was applied as the agonist for AMPA receptors. Thiopental (3-300 microM), R(-)-MPPB or S(+)-MPPB (100-1,000 microM) was coapplied with kainate under the condition in which the GABA(A) receptor-mediated current was blocked. Effects of MPPB isomers on the current elicited by GABA 1 microM were studied in the separate experiments. Results Thiopental inhibited the kainate-induced current reversibly and in a dose-dependent manner, with a concentration for 50% inhibition of 49.3 microM. Both R(-)-MPPB and S(+)-MPPB inhibited the kainate-induced current with a little stereoselectivity. R(-)-MPPB was slightly but significantly more potent than S(+)-MPPB. In contrast, R(-)-MPPB enhanced but S(+)-MPPB reduced the GABA-induced current. Conclusions Both convulsant and depressant stereoisomers of the barbiturate inhibited the AMPA receptor-mediated current despite of their opposite effects on the central nervous system in vivo. Although thiopental exhibited a considerable inhibition of AMPA receptors, the results suggest that the inhibition of AMPA receptors contributes little to the hypnotic action of the barbiturates.

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The Role of Loop 5 in Acetylcholine Receptor Channel Gating

The Journal of General Physiology ◽

10.1085/jgp.200308885 ◽

2003 ◽

Vol 122 (5) ◽

pp. 521-539 ◽

Cited By ~ 51

Author(s):

Sudha Chakrapani ◽

Timothy D. Bailey ◽

Anthony Auerbach

Keyword(s):

Binding Site ◽

Acetylcholine Receptor ◽

Single Channel ◽

Channel Gating ◽

Α Subunit ◽

Acetylcholine Binding Protein ◽

Relationship Analysis ◽

Subunit Interface ◽

Acetylcholine Receptor Channel ◽

Receptor Channel

Nicotinic acetylcholine receptor channel (AChR) gating is an organized sequence of molecular motions that couples a change in the affinity for ligands at the two transmitter binding sites with a change in the ionic conductance of the pore. Loop 5 (L5) is a nine-residue segment (mouse α-subunit 92–100) that links the β4 and β5 strands of the extracellular domain and that (in the α-subunit) contains binding segment A. Based on the structure of the acetylcholine binding protein, we speculate that in AChRs L5 projects from the transmitter binding site toward the membrane along a subunit interface. We used single-channel kinetics to quantify the effects of mutations to αD97 and other L5 residues with respect to agonist binding (to both open and closed AChRs), channel gating (for both unliganded and fully-liganded AChRs), and desensitization. Most αD97 mutations increase gating (up to 168-fold) but have little or no effect on ligand binding or desensitization. Rate-equilibrium free energy relationship analysis indicates that αD97 moves early in the gating reaction, in synchrony with the movement of the transmitter binding site (Φ = 0.93, which implies an open-like character at the transition state). αD97 mutations in the two α-subunits have unequal energetic consequences for gating, but their contributions are independent. We conclude that the key, underlying functional consequence of αD97 perturbations is to increase the unliganded gating equilibrium constant. L5 emerges as an important and early link in the AChR gating reaction which, in the absence of agonist, serves to increase the relative stability of the closed conformation of the protein.

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Low pH Potentiates Both Capsaicin Binding and Channel Gating of VR1 Receptors

The Journal of General Physiology ◽

10.1085/jgp.200308847 ◽

2003 ◽

Vol 122 (1) ◽

pp. 45-61 ◽

Cited By ~ 67

Author(s):

Sujung Ryu ◽

Beiying Liu ◽

Feng Qin

Keyword(s):

Single Channel ◽

Channel Gating ◽

General Mechanism ◽

Low Concentrations ◽

Excised Patches ◽

High Concentrations ◽

Activation Pathways ◽

Single Channel Currents ◽

Channel Currents ◽

Important Basis

Capsaicin ion channels are highly expressed in peripheral nervous terminals and involved in pain and thermal sensations. One characteristic of the cloned VR1 receptor is its multimodal responses to various types of noxious stimuli. The channel is independently activated by capsaicin and related vanilloids at submicromolar range, by heat above 40°C, and by protons at pH below 6.5. Furthermore, simultaneous applications of two or more stimuli lead to cross sensitization of the receptor, with an apparent increase in the sensitivity to any individual stimulus when applied alone. We studied here the mechanism underlying such cross-sensitization; in particular, between capsaicin and pH, two prototypical stimuli for the channel. By analyzing single-channel currents recorded from excised-patches expressing single recombinant VR1 receptors, we examined the effect of pH on burst properties of capsaicin activation at low concentrations and the effect on gating kinetics at high concentrations. Our results indicate that pH has dual effects on both capsaicin binding and channel gating. Lowering pH enhances the apparent binding affinity of capsaicin, promotes the occurrences of long openings and short closures, and stabilizes at least one of the open conformations of the channel. Our data also demonstrate that capsaicin binding and protonation of the receptor interact allosterically, where the effect of one can be offset by the effect of the other. These results provide important basis to further understand the nature of the activation pathways of the channel evoked by different stimuli as well as the general mechanism underling the cross-sensitization of pain.

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Functional Roles of Nonconserved Structural Segments in CFTR's NH2-terminal Nucleotide Binding Domain

The Journal of General Physiology ◽

10.1085/jgp.200409174 ◽

2004 ◽

Vol 125 (1) ◽

pp. 43-55 ◽

Cited By ~ 41

Author(s):

László Csanády ◽

Kim W. Chan ◽

Angus C. Nairn ◽

David C. Gadsby

Keyword(s):

Cystic Fibrosis ◽

Conformational Changes ◽

Single Channel ◽

Channel Gating ◽

Essential Element ◽

Nucleotide Binding ◽

Transmembrane Conductance Regulator ◽

Functional Roles ◽

Binding Domains ◽

Excised Patches

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282–293) identified two regions absent from structures of all other NBDs determined so far, a “regulatory insertion” (residues 404–435) and a “regulatory extension” (residues 639–670), both positioned to impede formation of the putative NBD1–NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1–414 with residues 433–1480, or residues 1–633 with 668–1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.

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Faculty Opinions recommendation of PICK1 mediates transient synaptic expression of GluA2-lacking AMPA receptors during glycine-induced AMPA receptor trafficking.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717970994.793469387 ◽

2013 ◽

Author(s):

Johannes Hell

Keyword(s):

Ampa Receptor ◽

Ampa Receptors ◽

Receptor Trafficking ◽

Ampa Receptor Trafficking

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AMPA Receptor Antagonist CFM-2 Decreases Survivin Expression in Cancer Cells

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180228123406 ◽

2018 ◽

Vol 18 (4) ◽

pp. 591-596 ◽

Cited By ~ 1

Author(s):

Domingo Sanchez Ruiz ◽

Hella Luksch ◽

Marco Sifringer ◽

Achim Temme ◽

Christian Staufner ◽

...

Keyword(s):

Cell Survival ◽

Receptor Antagonist ◽

Cancer Cells ◽

Glutamate Receptors ◽

Cancer Cell ◽

Ampa Receptor ◽

Ampa Receptors ◽

Survivin Expression ◽

Ampa Receptor Antagonist ◽

Cancer Cell Survival

Background: Glutamate receptors are widely expressed in different types of cancer cells. α-Amino-3- hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are ionotropic glutamate receptors which are coupled to intracellular signaling pathways that influence cancer cell survival, proliferation, and migration. Blockade of AMPA receptors by pharmacologic compounds may potentially constitute an effective tool in anticancer treatment strategies. Method: Here we investigated the impact of the AMPA receptor antagonist CFM-2 on the expression of the protein survivin, which is known to promote cancer cell survival and proliferation. We show that CFM-2 inhibits survivin expression at mRNA and protein levels and decreases the viability of cancer cells. Using a stably transfected cell line which overexpresses survivin, we demonstrate that over-expression of survivin enhances cancer cell viability and attenuates CFM-2–mediated inhibition of cancer cell growth. Result: These findings point towards suppression of survivin expression as a new mechanism contributing to anticancer effects of AMPA antagonists.

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AMPA and Kainate Receptors

The Oxford Handbook of Neuronal Ion Channels ◽

10.1093/oxfordhb/9780190669164.013.8 ◽

2020 ◽

Author(s):

G. Brent Dawe ◽

Patricia M. G. E. Brown ◽

Derek Bowie

Keyword(s):

Ion Channel ◽

Glutamate Receptor ◽

Single Channel ◽

Channel Gating ◽

Regulatory Proteins ◽

Kainate Receptors ◽

Mammalian Brain ◽

Receptor Field ◽

Neuronal Excitation ◽

Ion Channel Proteins

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate-type glutamate receptors (AMPARs and KARs) are dynamic ion channel proteins that govern neuronal excitation and signal transduction in the mammalian brain. The four AMPAR and five KAR subunits can heteromerize with other subfamily members to create several combinations of tetrameric channels with unique physiological and pharmacological properties. While both receptor classes are noted for their rapid, millisecond-scale channel gating in response to agonist binding, the intricate structural rearrangements underlying their function have only recently been elucidated. This chapter begins with a review of AMPAR and KAR nomenclature, topology, and rules of assembly. Subsequently, receptor gating properties are outlined for both single-channel and synaptic contexts. The structural biology of AMPAR and KAR proteins is also discussed at length, with particular focus on the ligand-binding domain, where allosteric regulation and alternative splicing work together to dictate gating behavior. Toward the end of the chapter there is an overview of several classes of auxiliary subunits, notably transmembrane AMPAR regulatory proteins and Neto proteins, which enhance native AMPAR and KAR expression and channel gating, respectively. Whether bringing an ion channel novice up to speed with glutamate receptor theory and terminology or providing a refresher for more seasoned biophysicists, there is much to appreciate in this summation of work from the glutamate receptor field.

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