Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation

Most ligand- and voltage-gated ion channels assemble as signaling complexes consisting of pore-forming and auxiliary subunits. In the mammalian brain, AMPA-type ionotropic glutamate receptors (AMPARs) coassemble with several families of auxiliary subunits that regulate channel gating as well as ion channel block and permeation. Previous work has shown that auxiliary proteins stargazin (or γ2) and cornichon-3 (CNIH-3) attenuate the cytoplasmic polyamine channel block of AMPARs, although the underlying mechanism has yet to be established. Here, we show that γ2 and CNIH-3 relieve channel block by enhancing the rate of blocker permeation. Surprisingly, the relative permeability of the polyamine spermine (Spm) through the pore of the AMPAR-γ2 or -CNIH-3 complexes is considerably more than AMPARs expressed alone. Spm permeability is comparable to that of Na+ for the GluA2-γ2 complex and four times greater than Na+ with GluA2 + CNIH-3. A modified model of permeant channel block fully accounts for both the voltage- and time-dependent nature of Spm block. Estimates of block rate constants reveal that auxiliary subunits do not attenuate block by shifting the location of the block site within the membrane electric field, and they do not affect the blocker’s ability to reach it. Instead, γ2 and CNIH-3 relieve channel block by facilitating the blocker’s exit rates from the open channel. From a physiological perspective, the relief of channel block exerted by γ2 and CNIH-3 ensures that there is unfettered signaling by AMPARs at glutamatergic synapses. Moreover, the pronounced ability of AMPARs to transport polyamines may have an unexpected role in regulating cellular polyamine levels.

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Modulation of AMPA receptor function by auxiliary subunits

e-Neuroforum ◽

10.1515/s13295-015-0005-z ◽

2015 ◽

Vol 21 (2) ◽

Author(s):

Hannah Monyer ◽

Jakob von Engelhardt

Keyword(s):

Ampa Receptors ◽

Ionotropic Glutamate Receptors ◽

Receptor Function ◽

Short Term ◽

Auxiliary Subunits ◽

Excitatory Transmission ◽

Receptor Localization ◽

Physiological Properties ◽

The Central Nervous System

AbstractAMPA receptors are ionotropic glutamate receptors that mediate the majority of fast excitatory transmission in the central nervous system. Their function depends not only on the composition of the subunits GluA1-4, but also on the interaction with auxiliary subunits. Several auxiliary subunits have been identified in proteomic analyses over the last years and we are beginning to understand the complex control of these proteins on physiological properties and membrane- transport of AMPA receptors. Auxiliary subunits such as TARPs, cornichons, and CK-AMP44 influence receptor localization on the cell membrane, modulate receptor gating, and play a role for synaptic short-term and long-term plasticity.

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Auxiliary subunits keep AMPA receptors compact during activation and desensitization

10.1101/295105 ◽

2018 ◽

Author(s):

Jelena Baranovic ◽

Andrew J.R. Plested

Keyword(s):

Ampa Receptors ◽

Conformational Flexibility ◽

Ionotropic Glutamate Receptors ◽

Excitatory Synapses ◽

Auxiliary Subunits ◽

Auxiliary Subunit ◽

Cryo Electron Microscopy ◽

Wide Range ◽

Technical Advances ◽

Conformational Freedom

SummarySignal transduction at vertebrate excitatory synapses involves the activity of ionotropic glutamate receptors, including the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor. Technical advances in cryo-electron microscopy have brought a slew of full-length structures of AMPA receptors, on their own and in combination with auxiliary subunits. These structures illustrate a wide range of conformations, indicating that individual domains might undergo substantial lateral motions during gating, resulting in an open, “relaxed” extracellular layer. Here, we used bifunctional methanethiosulfonate cross-linkers to calibrate the conformations found in functional AMPA receptors both in the presence and absence of the auxiliary subunit Stargazin. Our data indicate that AMPA receptors have considerable conformational freedom and can get trapped in stable, relaxed conformations, especially upon long exposures to glutamate. In contrast, Stargazin limits this conformational flexibility. Thus, under synaptic conditions, where brief glutamate exposures and the presence of Stargazin dominate, AMPA receptors are unlikely to adopt very relaxed conformations during gating.

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Open-channel-block induced by the neurotransmitter glycine in homomeric alpha1-GlyR and heteromeric alpha1/beta1- GlyR

Aktuelle Neurologie ◽

10.1055/s-0028-1086720 ◽

2008 ◽

Vol 35 (S 01) ◽

Author(s):

Y.P Song ◽

F Schlesinger ◽

S Petri ◽

R Dengler ◽

K Krampfl

Keyword(s):

Open Channel ◽

Channel Block ◽

Open Channel Block

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Pharmacological modulation of AMPA receptors rescues specific impairments in social behavior associated with the A350V Iqsec2 mutation

Translational Psychiatry ◽

10.1038/s41398-021-01347-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Renad Jabarin ◽

Nina Levy ◽

Yasmin Abergel ◽

Joshua H. Berman ◽

Amir Zag ◽

...

Keyword(s):

Social Behavior ◽

Ampa Receptors ◽

Emotional State ◽

Social Discrimination ◽

Glutamatergic Synapses ◽

Behavioral Deficits ◽

Pharmacological Modulation ◽

The Social ◽

State Preference ◽

Molecular Pathophysiology

AbstractIn this study we tested the hypothesis that pharmacological modulation of glutamatergic neurotransmission could rescue behavioral deficits exhibited by mice carrying a specific mutation in the Iqsec2 gene. The IQSEC2 protein plays a key role in glutamatergic synapses and mutations in the IQSEC2 gene are a frequent cause of neurodevelopmental disorders. We have recently reported on the molecular pathophysiology of one such mutation A350V and demonstrated that this mutation downregulates AMPA type glutamatergic receptors (AMPAR) in A350V mice. Here we sought to identify behavioral deficits in A350V mice and hypothesized that we could rescue these deficits by PF-4778574, a positive AMPAR modulator. Using a battery of social behavioral tasks, we found that A350V Iqsec2 mice exhibit specific deficits in sex preference and emotional state preference behaviors as well as in vocalizations when encountering a female mouse. The social discrimination deficits, but not the impaired vocalization, were rescued with a single dose of PF-4778574. We conclude that social behavior deficits associated with the A350V Iqsec2 mutation may be rescued by enhancing AMPAR mediated synaptic transmission.

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Control of AMPA receptor activity by the extracellular loops of auxiliary proteins

eLife ◽

10.7554/elife.28680 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 15

Author(s):

Irene Riva ◽

Clarissa Eibl ◽

Rudolf Volkmer ◽

Anna L Carbone ◽

Andrew JR Plested

Keyword(s):

Ampa Receptor ◽

Ampa Receptors ◽

De Novo ◽

Dominant Role ◽

Mammalian Brain ◽

Binding Assays ◽

Receptor Kinetics ◽

Receptor Complexes ◽

Extracellular Loops ◽

Kinetics Of

At synapses throughout the mammalian brain, AMPA receptors form complexes with auxiliary proteins, including TARPs. However, how TARPs modulate AMPA receptor gating remains poorly understood. We built structural models of TARP-AMPA receptor complexes for TARPs γ2 and γ8, combining recent structural studies and de novo structure predictions. These models, combined with peptide binding assays, provide evidence for multiple interactions between GluA2 and variable extracellular loops of TARPs. Substitutions and deletions of these loops had surprisingly rich effects on the kinetics of glutamate-activated currents, without any effect on assembly. Critically, by altering the two interacting loops of γ2 and γ8, we could entirely remove all allosteric modulation of GluA2, without affecting formation of AMPA receptor-TARP complexes. Likewise, substitutions in the linker domains of GluA2 completely removed any effect of γ2 on receptor kinetics, indicating a dominant role for this previously overlooked site proximal to the AMPA receptor channel gate.

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Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423205112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6176-6181 ◽

Cited By ~ 61

Author(s):

Olga Varea ◽

Maria Dolores Martin-de-Saavedra ◽

Katherine J. Kopeikina ◽

Britta Schürmann ◽

Hunter J. Fleming ◽

...

Keyword(s):

Ampa Receptors ◽

Knockout Mice ◽

Copy Number Variations ◽

Spine Density ◽

Structured Illumination ◽

Single Nucleotide Variants ◽

Glutamatergic Synapses ◽

Superresolution Microscopy ◽

Synaptic Markers ◽

Cellular Phenotypes

Central glutamatergic synapses and the molecular pathways that control them are emerging as common substrates in the pathogenesis of mental disorders. Genetic variation in the contactin associated protein-like 2 (CNTNAP2) gene, including copy number variations, exon deletions, truncations, single nucleotide variants, and polymorphisms have been associated with intellectual disability, epilepsy, schizophrenia, language disorders, and autism. CNTNAP2, encoded by Cntnap2, is required for dendritic spine development and its absence causes disease-related phenotypes in mice. However, the mechanisms whereby CNTNAP2 regulates glutamatergic synapses are not known, and cellular phenotypes have not been investigated in Cntnap2 knockout neurons. Here we show that CNTNAP2 is present in dendritic spines, as well as axons and soma. Structured illumination superresolution microscopy reveals closer proximity to excitatory, rather than inhibitory synaptic markers. CNTNAP2 does not promote the formation of synapses and cultured neurons from Cntnap2 knockout mice do not show early defects in axon and dendrite outgrowth, suggesting that CNTNAP2 is not required at this stage. However, mature neurons from knockout mice show reduced spine density and levels of GluA1 subunits of AMPA receptors in spines. Unexpectedly, knockout neurons show large cytoplasmic aggregates of GluA1. Here we characterize, for the first time to our knowledge, synaptic phenotypes in Cntnap2 knockout neurons and reveal a novel role for CNTNAP2 in GluA1 trafficking. Taken together, our findings provide insight into the biological roles of CNTNAP2 and into the pathogenesis of CNTNAP2-associated neuropsychiatric disorders.

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Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

The Journal of General Physiology ◽

10.1085/jgp.201310984 ◽

2013 ◽

Vol 142 (3) ◽

pp. 191-206 ◽

Cited By ~ 16

Author(s):

Amanda H. Lewis ◽

Indira M. Raman

Keyword(s):

Open Channel ◽

Voltage Sensor ◽

Channel Block ◽

Na Channels ◽

Fast Inactivation ◽

Na Current ◽

Open Channel Block ◽

Channel Blocking ◽

Open States ◽

Resurgent Currents

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

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