ABSTRACT
Preclinical assessments of pain have often relied upon behavioral measurements and anesthetized neurophysiological recordings. Current technologies enabling large-scale neural recordings, however, have the potential to unveil quantifiable pain signals in conscious animals for preclinical studies. Although pain processing is distributed across many brain regions, the anterior cingulate cortex (ACC) is of particular interest in isolating these signals given its suggested role in the affective ("unpleasant") component of pain. Here, we explored the utility of the ACC toward preclinical pain research using head-mounted miniaturized microscopes to record calcium transients in freely moving male mice expressing genetically encoded calcium indicator 6f (GCaMP6f) under the Thy1 promoter. We verified the expression of GCaMP6f in excitatory neurons and found no intrinsic behavioral differences in this model. Using a multimodal stimulation paradigm across naive, pain, and analgesic conditions, we found that while ACC population activity roughly scaled with stimulus intensity, single-cell representations were highly flexible. We found only low-magnitude increases in population activity after complete Freund's adjuvant (CFA) and insufficient evidence for the existence of a robust nociceptive ensemble in the ACC. However, we found a temporal sharpening of response durations and generalized increases in pairwise neural correlations in the presence of the mechanistically distinct analgesics gabapentin or ibuprofen after (but not before) CFA-induced inflammatory pain. This increase was not explainable by changes in locomotion alone. Taken together, these results highlight challenges in isolating distinct pain signals among flexible representations in the ACC but suggest a neurophysiological hallmark of analgesia after pain that generalizes to at least two analgesics.
Subject(s)
Gyrus Cinguli , Animals , Mice , Male , Gyrus Cinguli/physiopathology , Gyrus Cinguli/drug effects , Pain/physiopathology , Inflammation , Mice, Inbred C57BL , Analgesia/methods , Analgesics/pharmacology , Freund's Adjuvant/toxicity , Ibuprofen/pharmacologyABSTRACT
Nonselective glutamate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonists are efficacious in chronic pain but have significant tolerability issues, likely arising from the ubiquitous expression of AMPA receptors in the central nervous system (CNS). Recently, LY3130481 has been shown to selectively block AMPA receptors coassembled with the auxiliary protein, transmembrane AMPA receptor regulatory protein (TARP) γ8, which is highly expressed in the hippocampus but also in pain pathways, including anterior cingulate (ACC) and somatosensory cortices and the spinal cord, suggesting that selective blockade of γ8/AMPA receptors may suppress nociceptive signaling with fewer CNS side effects. The potency of LY3130481 on recombinant γ8-containing AMPA receptors was modulated by coexpression with other TARPs; γ2 subunits affected activity more than γ3 subunits. Consistent with these findings, LY3130481 had decreasing potency on receptors from rat hippocampal, cortical, spinal cord, and cerebellar neurons that was replicated in tissue from human brain. LY3130481 partially suppressed, whereas the nonselective AMPA antagonist GYKI53784 completely blocked, AMPA receptor-dependent excitatory postsynaptic potentials in ACC and spinal neurons in vitro. Similarly, LY3130481 attenuated short-term synaptic plasticity in spinal sensory neurons in vivo in response to stimulation of peripheral afferents. LY3130481 also significantly reduced nocifensive behaviors after intraplantar formalin that was correlated with occupancy of CNS γ8-containing AMPA receptors. In addition, LY3130481 dose-dependently attenuated established gait impairment after joint damage and tactile allodynia after spinal nerve ligation, all in the absence of motor side effects. Collectively, these data demonstrate that LY3130481 can suppress excitatory synaptic transmission and plasticity in pain pathways containing γ8/AMPA receptors and significantly reduce nocifensive behaviors, suggesting a novel, effective, and safer therapy for chronic pain conditions.
Subject(s)
Calcium Channels/metabolism , Chronic Pain/drug therapy , Chronic Pain/metabolism , Molecular Targeted Therapy , Receptors, AMPA/metabolism , Animals , Benzothiazoles/pharmacology , Benzothiazoles/therapeutic use , Chronic Pain/physiopathology , Male , Neuronal Plasticity/drug effects , Nociception/drug effects , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Rats , Rats, Sprague-Dawley , Synaptic Transmission/drug effects , Tissue DistributionABSTRACT
The need for improved medications for psychiatric and neurological disorders is clear. Difficulties in finding such drugs demands that all strategic means be utilized for their invention. The discovery of forebrain specific AMPA receptor antagonists, which selectively block the specific combinations of principal and auxiliary subunits present in forebrain regions but spare targets in the cerebellum, was recently disclosed. This discovery raised the possibility that other auxiliary protein systems could be utilized to help identify new medicines. Discussion of the TARP-dependent AMPA receptor antagonists has been presented elsewhere. Here we review the diversity of protein complexes of neurotransmitter receptors in the nervous system to highlight the broad range of protein/protein drug targets. We briefly outline the structural basis of protein complexes as drug targets for G-protein-coupled receptors, voltage-gated ion channels, and ligand-gated ion channels. This review highlights heterodimers, subunit-specific receptor constructions, multiple signaling pathways, and auxiliary proteins with an emphasis on the later. We conclude that the use of auxiliary proteins in chemical compound screening could enhance the detection of specific, targeted drug searches and lead to novel and improved medicines for psychiatric and neurological disorders.
Subject(s)
Calcium Channels/metabolism , Drug Discovery , Mental Disorders/drug therapy , Nervous System Diseases/drug therapy , Protein Multimerization , Receptors, AMPA/antagonists & inhibitors , Animals , Benzothiazoles/therapeutic use , Humans , Mental Disorders/metabolism , Molecular Targeted Therapy , Nervous System Diseases/metabolism , Protein Subunits , Pyrazoles/therapeutic useABSTRACT
Drugs originate from the discovery of compounds, natural or synthetic, that bind to proteins (receptors, enzymes, transporters, etc.), the interaction of which modulates biological cascades that have potential therapeutic benefit. Rational strategies for identifying novel drug therapies are typically based on knowledge of the structure of the target proteins and the design of new chemical entities that modulate these proteins in a beneficial manner. The present review discusses a novel approach to drug discovery based on the identification and characterization of auxiliary proteins, the transmembrane AMPA receptor regulatory proteins (TARPs) that are associated with AMPA receptors. Utilizing these auxiliary proteins in compound screening led to the discovery of the TARP-dependent-AMPA forebrain selective receptor antagonist (TDAA), LY3130481/CERC-611 that is currently in clinical development for epilepsy.
Subject(s)
Benzothiazoles/pharmacology , Drug Discovery/methods , Prosencephalon/drug effects , Prosencephalon/metabolism , Pyrazoles/pharmacology , Receptors, AMPA/antagonists & inhibitors , Receptors, AMPA/metabolism , Animals , Benzothiazoles/chemistry , Humans , Protein Subunits/antagonists & inhibitors , Protein Subunits/metabolism , Pyrazoles/chemistryABSTRACT
BACKGROUND & OBJECTIVE: 6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]- 3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8 that is under development for epilepsy. The present study provided a broad inquiry into its anticonvulsant properties. LY3130481 was anticonvulsant in multiple acute seizure provocation models in mice and rats. In addition, LY3130481 was effective against absence seizures in the GAERS genetic model and in the Frings mouse model. Likewise, LY3130481 attenuated convulsions in mice and rats with long-term induction of seizures (e.g., corneal, pentylenetetrazole, hippocampal, and amygdala kindled seizures). In slices of epileptic human cortex, LY3130481 significantly decreased neuronal firing frequencies. LY3130481 displaced from rat brain a radioligand specific for AMPA receptors associated with TARP γ-8 whereas non-TARP-selective molecules did not. Binding was also observed in hippocampus freshly transected from a patient. RESULTS & CONCLUSION: Taken as a whole, the findings reported here establish the broad anticonvulsant efficacy of LY3130481 indicating that blockade of AMPA receptors associated with TARP γ-8 is sufficient for these protective effects.
Subject(s)
Benzothiazoles/pharmacology , Calcium Channels/metabolism , Pyrazoles/pharmacology , Receptors, AMPA/antagonists & inhibitors , Seizures/prevention & control , Animals , Anticonvulsants/pharmacology , Cerebral Cortex/physiology , Disease Models, Animal , Female , Humans , Male , Mice , Neurons/physiology , Radioligand Assay , RatsABSTRACT
6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]-3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food-maintained responding in rats and spontaneous alternation in a Y-maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal-dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive-impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor-impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non-rapid-eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro-cognitive and wake-promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5-HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC-611 as an antiepileptic.
Subject(s)
Anticonvulsants/administration & dosage , Benzothiazoles/administration & dosage , Calcium Channels/physiology , Cognition/drug effects , Prefrontal Cortex/drug effects , Pyrazoles/administration & dosage , Acetylcholine/metabolism , Animals , Behavior, Animal/drug effects , Conditioning, Classical/drug effects , Electroencephalography , Fear/drug effects , Fructose/administration & dosage , Fructose/analogs & derivatives , Histamine/metabolism , Male , Maze Learning/drug effects , Nitriles , Prefrontal Cortex/metabolism , Prefrontal Cortex/physiology , Pyridones/administration & dosage , Rats, Sprague-Dawley , Rats, Wistar , Serotonin/metabolism , Sleep Stages/drug effects , TopiramateABSTRACT
The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.
Subject(s)
Benzothiazoles/chemistry , Molecular Docking Simulation , Neurons/chemistry , Pyrazoles/chemistry , Receptors, AMPA/antagonists & inhibitors , Receptors, AMPA/ultrastructure , Animals , Binding Sites , Hippocampus/chemistry , Male , Mice , Models, Biological , Models, Chemical , Protein Binding , Protein Conformation , Structure-Activity RelationshipABSTRACT
Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.
Subject(s)
Anticonvulsants/pharmacology , Benzothiazoles/pharmacology , Cerebellum/drug effects , Epilepsy/drug therapy , Prosencephalon/drug effects , Pyrazoles/pharmacology , Pyridones/pharmacology , Receptors, AMPA/antagonists & inhibitors , Animals , Anticonvulsants/adverse effects , Calcium Channels/metabolism , Cerebellum/metabolism , Convulsants/toxicity , Disease Models, Animal , Dizziness/chemically induced , Epilepsy/chemically induced , Mice , Nitriles , Pentylenetetrazole/toxicity , Prosencephalon/metabolism , Pyridones/adverse effects , Rats , Receptors, AMPA/metabolism , Seizures/chemically induced , Seizures/drug therapyABSTRACT
Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.
Subject(s)
Calcium Channels/metabolism , Drug Discovery , Receptors, AMPA/antagonists & inhibitors , High-Throughput Screening Assays , Humans , Molecular Docking Simulation , Molecular Structure , Receptors, AMPA/metabolismABSTRACT
Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber-Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.
Subject(s)
Neurons/physiology , Protein Kinase C/physiology , Purkinje Cells/physiology , Receptors, Glutamate/physiology , Receptors, Metabotropic Glutamate/physiology , Synaptic Transmission/physiology , TRPC Cation Channels/physiology , Animals , Excitatory Postsynaptic Potentials/genetics , Excitatory Postsynaptic Potentials/physiology , Female , Immunohistochemistry , Male , Mice , Mice, Knockout , Mutation/physiology , Patch-Clamp Techniques , Phenotype , Receptors, Cell Surface/physiology , Receptors, Glutamate/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Solubility , Subcellular Fractions/metabolism , Subcellular Fractions/physiologyABSTRACT
Transmembrane AMPA receptor regulatory proteins (TARPs) are auxiliary subunits that modulate AMPA receptor trafficking, gating and pharmacology throughout the brain. Why cornichon-2 (CNIH-2), another AMPA receptor-associated protein, modulates AMPA receptor gating and pharmacology in hippocampal neurons but not cerebellar granule neurons remains unresolved. Here, we report that CNIH-2 differentially impacts Type-Ia (γ-2 or γ-3) vs. Type-Ib (γ-4 or γ-8) TARP-containing AMPA receptors. Specifically, with AMPA receptors comprising γ-2, the cerebellar-enriched TARP isoform, CNIH-2 decreases I(KA) /I(Glu) ratio and decreases cyclothiazide efficacy while having minimal impact on recovery from desensitization and deactivation kinetics. By contrast, with AMPA receptors comprising γ-8, the hippocampal-enriched TARP isoform, we find that CNIH-2 slows deactivation kinetics, increases cyclothiazide potency and occludes a novel AMPA receptor kinetic phenomenon, namely resensitization. Additionally, we find that CNIH-2 differentially modulates the glutamate off-kinetics of γ-8-containing, but not γ-2-containing, AMPA receptors in a manner dependent upon the duration of agonist application. Together, these data demonstrate that the modulation of AMPA receptors by CNIH-2 depends upon the TARP isoform composition within the receptor complex.
Subject(s)
Brain/metabolism , Egg Proteins/metabolism , Membrane Proteins/metabolism , Neurons/metabolism , Receptors, AMPA/metabolism , HEK293 Cells , Humans , Ion Channel Gating/physiology , Patch-Clamp Techniques , Protein Isoforms/metabolism , TransfectionABSTRACT
The reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPA receptor (AMPAR) auxiliary subunit TARPγ-8 could be a result of a reduction in AMPAR expression or of the direct action of γ-8. We generated TARPγ-8Δ4 knock-in mice lacking the C-terminal PDZ ligand. We found that synaptic transmission and AMPARs were reduced in the mutant mice, but extrasynaptic AMPAR expression and long-term potentiation (LTP) were unaltered. Our findings suggest that there are distinct TARP-dependent mechanisms for synaptic transmission and LTP.
Subject(s)
Membrane Proteins/metabolism , Neuronal Plasticity/physiology , PDZ Domains/physiology , Synaptic Transmission/physiology , Age Factors , Animals , Animals, Newborn , Biophysics , Calcium Channels/genetics , Disks Large Homolog 4 Protein , Electric Stimulation , Gene Expression Regulation, Developmental/genetics , Guanylate Kinases/metabolism , Hippocampus/cytology , Hippocampus/drug effects , In Vitro Techniques , Long-Term Potentiation/drug effects , Long-Term Potentiation/genetics , Membrane Proteins/genetics , Mice , Mice, Transgenic , Models, Biological , Mutation/genetics , Neuronal Plasticity/genetics , PDZ Domains/genetics , Patch-Clamp Techniques , Synaptic Transmission/genetics , Synaptophysin/metabolism , Synaptosomes/metabolismABSTRACT
Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon-2/3 (CNIH-2/3). The mechanisms that control AMPA receptor complex assembly remain unclear. AMPA receptor responses in neurons differ from those in cell lines transfected with GluA plus TARPs γ-8 or γ-7, which show unusual resensitization kinetics and non-native AMPA receptor pharmacologies. Using tandem GluA/TARP constructs to constrain stoichiometry, we show here that these peculiar kinetic and pharmacological signatures occur in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuron-like kinetics and pharmacologies, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that coexpression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface in a γ-8-dependent manner to dictate receptor pharmacology. In the cerebellum, however, CNIH-2 expressed in Purkinje neurons does not reach the neuronal surface. In concordance, stargazer Purkinje neurons, which express CNIH-2 and γ-7, display AMPA receptor kinetics/pharmacologies that can only be recapitulated recombinantly by a low γ-7/GluA stoichiometry. Together, these data suggest that CNIH-2 modulates neuronal AMPA receptor auxiliary subunit assembly by regulating the number of TARPs within an AMPA receptor complex to modulate receptor gating and pharmacology.
Subject(s)
Ion Channel Gating/physiology , Membrane Proteins/metabolism , Neurons/metabolism , Protein Subunits/metabolism , Receptors, AMPA/metabolism , Analysis of Variance , Animals , Cell Membrane/metabolism , Cells, Cultured , Cerebellar Cortex/cytology , Cerebellar Cortex/metabolism , Electrophysiology , Hippocampus/cytology , Hippocampus/metabolism , Male , Mice , Neurons/cytology , Rats , Rats, WistarABSTRACT
AMPA receptors mediate fast excitatory transmission in the brain. Neuronal AMPA receptors comprise GluA pore-forming principal subunits and can associate with multiple modulatory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and CNIHs (cornichons). AMPA receptor potentiators and non-competitive antagonists represent potential targets for a variety of neuropsychiatric disorders. Previous studies showed that the AMPA receptor antagonist GYKI-53655 displaces binding of a potentiator from brain receptors but not from recombinant GluA subunits. Here, we asked whether AMPA receptor modulatory subunits might resolve this discrepancy. We find that the cerebellar TARP, stargazin (γ-2), enhances the binding affinity of the AMPA receptor potentiator [(3)H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [(3)H]-LY450295 binding is reduced and relatively resistant to displacement by non-competitive antagonists. Coexpression of AMPA receptors with CNIH-2, which is expressed in the hippocampus and at low levels in the cerebellar Purkinje neurons, confers partial sensitivity of [(3)H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [(3)H]-LY450295 binding to stargazer and γ-8-deficient mouse brain sections, demonstrates that TARPs regulate the pharmacology of allosteric AMPA potentiators and antagonists in the cerebellum and hippocampus, respectively. These studies demonstrate that accessory proteins define AMPA receptor pharmacology by functionally linking allosteric AMPA receptor potentiator and antagonist sites.
Subject(s)
Benzodiazepines/pharmacology , Cell Membrane/metabolism , Egg Proteins/metabolism , Excitatory Amino Acid Antagonists/pharmacology , Membrane Proteins/metabolism , Purkinje Cells/metabolism , Receptors, AMPA , Allosteric Regulation/genetics , Animals , Calcium Channels/genetics , Calcium Channels/metabolism , Cell Membrane/genetics , Egg Proteins/genetics , HEK293 Cells , Humans , Membrane Proteins/genetics , Mice , Receptors, AMPA/agonists , Receptors, AMPA/antagonists & inhibitors , Receptors, AMPA/genetics , Receptors, AMPA/metabolismABSTRACT
Transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon proteins (CNIH-2/3) independently modulate AMPA receptor trafficking and gating. However, the potential for interactions of these subunits within an AMPA receptor complex is unknown. Here, we find that TARPs γ-4, γ-7, and γ-8, but not γ-2, γ-3, or γ-5, cause AMPA receptors to "resensitize" upon continued glutamate application. With γ-8, resensitization occurs with all GluA subunit combinations; however, γ-8-containing hippocampal neurons do not display resensitization. In recombinant systems, CNIH-2 abrogates γ-8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match that of hippocampal neurons. In hippocampus, γ-8 and CNIH-2 associate in postsynaptic densities and CNIH-2 protein levels are markedly diminished in γ-8 knockout mice. Manipulating neuronal CNIH-2 levels modulates the electrophysiological properties of extrasynaptic and synaptic γ-8-containing AMPA receptors. Thus, γ-8 and CNIH-2 functionally interact with common hippocampal AMPA receptor complexes to modulate synergistically kinetics and pharmacology.
Subject(s)
Hippocampus/physiology , Ion Channel Gating/physiology , Membrane Proteins/physiology , Receptors, AMPA/physiology , Animals , Calcium Channels , Cells, Cultured , HEK293 Cells , Humans , Ion Channel Gating/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Rats , Rats, Wistar , Synaptic Potentials/genetics , Synaptic Potentials/physiologyABSTRACT
Transmembrane AMPA receptor regulatory proteins (TARPs) are the first identified auxiliary subunits for a neurotransmitter-gated ion channel. Although initial studies found that stargazin, the prototypical TARP, principally chaperones AMPA receptors, subsequent research demonstrated that it also regulates AMPA receptor kinetics and synaptic waveforms. Recent studies have identified a diverse collection of TARP isoforms--types Ia, Ib II--that distinctly regulate AMPA receptor trafficking, gating and neuropharmacology. These TARP isoforms are heterogeneously expressed in specific neuronal populations and can differentially sculpt synaptic transmission and plasticity. Whole-genome analyses also link multiple TARP loci to childhood epilepsy, schizophrenia and bipolar disorder. TARPs emerge as vital components of excitatory synapses that participate both in signal transduction and in neuropsychiatric disorders.
Subject(s)
Neuropharmacology , Nuclear Proteins/metabolism , Receptors, AMPA/metabolism , Animals , Central Nervous System Diseases/metabolism , Central Nervous System Diseases/pathology , Humans , Neuronal Plasticity/physiology , Neurons/physiology , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Synaptic Transmission/physiologyABSTRACT
AMPA-type glutamate receptors (GluRs) play major roles in excitatory synaptic transmission. Neuronal AMPA receptors comprise GluR subunits and transmembrane AMPA receptor regulatory proteins (TARPs). Previous studies identified five mammalian TARPs, gamma-2 (or stargazin), gamma-3, gamma-4, gamma-7, and gamma-8, that enhance AMPA receptor function. Here, we classify gamma-5 as a distinct class of TARP that modulates specific GluR2-containing AMPA receptors and displays properties entirely dissimilar from canonical TARPs. Gamma-5 increases peak currents and decreases the steady-state currents selectively from GluR2-containing AMPA receptors. Furthermore, gamma-5 increases rates of GluR2 deactivation and desensitization and decreases glutamate potency. Remarkably, all effects of gamma-5 require editing of GluR2 mRNA. Unlike other TARPs, gamma-5 modulates GluR2 without promoting receptor trafficking. We also find that gamma-7 regulation of GluR2 is dictated by mRNA editing. These data establish gamma-5 and gamma-7 as a separate family of "type II TARPs" that impart distinct physiological features to specific AMPA receptors.
Subject(s)
Ion Channel Gating/physiology , Membrane Potentials/physiology , Membrane Transport Proteins/metabolism , Neurons/metabolism , Receptors, AMPA/metabolism , Animals , Brain/cytology , Brain/metabolism , Cells, Cultured , Male , Membrane Proteins/metabolism , Protein Isoforms/metabolism , Rats , Rats, Sprague-Dawley , Rats, WistarABSTRACT
Ion channels link environmental stimuli to intracellular signaling pathways. Channel proteins often occur in macromolecular complexes in association with auxiliary subunits that control channel trafficking, gating and pharmacology. A large number of drugs exert their therapeutic effects by regulating ion channel activity and downstream signaling. These drugs can target either the principle ion channel or the associated auxiliary subunits. Sulfonylurea-type antidiabetics and gabapentin-type anticonvulsants exemplify important therapeutics that bind to ion channel auxiliary subunits. The recent molecular characterization of neuronal glutamate receptor ion channel complexes identified auxiliary subunits and associated proteins that may provide new targets for treating psychiatric and neurodegenerative diseases.
Subject(s)
Anticonvulsants/pharmacology , Drug Design , Hypoglycemic Agents/pharmacology , Ion Channels/metabolism , Animals , Anticonvulsants/chemistry , Humans , Hypoglycemic Agents/chemistry , Protein Binding , Protein SubunitsABSTRACT
AMPA-type glutamate receptors (GluRs) mediate most excitatory signaling in the brain and are composed of GluR principal subunits and transmembrane AMPA receptor regulatory protein (TARP) auxiliary subunits. Previous studies identified four mammalian TARPs, gamma-2 (or stargazin), gamma-3, gamma-4, and gamma-8, that control AMPA receptor trafficking, gating, and pharmacology. Here, we explore roles for the homologous gamma-5 and gamma-7 proteins, which were previously suggested not to serve as TARPs. Western blotting reveals high levels of gamma-5 and gamma-7 in the cerebellum, where gamma-7 is enriched in Purkinje neurons in the molecular layer and glomerular synapses in the granule cell layer. Immunoprecipitation proteomics shows that cerebellar gamma-7 avidly and selectively binds to AMPA receptor GluR subunits and also binds to the AMPA receptor clustering protein, postsynaptic density-95 (PSD-95). Furthermore, gamma-7 occurs together with PSD-95 and AMPA receptor subunits in purified postsynaptic densities. In heterologous cells, gamma-7 but not gamma-5 greatly enhances AMPA receptor glutamate-evoked currents and modulates channel gating. In granule cells from stargazer mice, transfection of gamma-7 but not gamma-5 increases AMPA receptor-mediated currents. Compared with stargazin, gamma-7 differentially modulates AMPA receptor glutamate affinity and kainate efficacy. These studies define gamma-7 as a new member of the TARP family that can differentially influence AMPA receptors in cerebellar neurons.