Group II metabotropic glutamate receptor (mGlu2 and mGlu3 ) roles in thalamic processing.

BACKGROUND AND PURPOSE: As the thalamus underpins almost all aspects of behaviour, it is important to understand how the thalamus operates. Group II metabotropic glutamate (mGlu2 /mGlu3 ) receptor activation reduces inhibition in thalamic nuclei originating from the surrounding thalamic reticular nucleus (TRN). Whilst an mGlu2 component to this effect has been reported, in this study, we demonstrate that it is likely, largely mediated via mGlu3 . EXPERIMENTAL APPROACH: The somatosensory ventrobasal thalamus (VB) is an established model for probing fundamental principles of thalamic function. In vitro slices conserving VB-TRN circuitry from wild-type and mGlu3 knockout mouse brains were used to record IPSPs and mIPSCs. In vivo extracellular recordings were made from VB neurons in anaesthetised rats. A range of selective pharmacological agents were used to probe Group II mGlu receptor function (agonist, LY354740; antagonist, LY341495; mGlu2 positive allosteric modulator, LY487379 and mixed mGlu2 agonist/mGlu3 antagonist LY395756). KEY RESULTS: The in vitro and in vivo data are complementary and suggest that mGlu3 receptor activation is largely responsible for potentiating responses to somatosensory stimulation by reducing inhibition from the TRN. CONCLUSIONS AND IMPLICATIONS: mGlu3 receptor activation in the VB likely enables important somatosensory information to be discerned from background activity. These mGlu3 receptors are likely to be endogenously activated via 'glutamate spillover'. In cognitive thalamic nuclei, this mechanism may be of importance in governing attentional processes. Positive allosteric modulation of endogenous mGlu3 receptor activation may therefore enhance cognitive function in pathophysiological disease states, such as schizophrenia, thus representing a highly specific therapeutic target. LINKED ARTICLES: This article is part of a themed issue on Building Bridges in Neuropharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.8/issuetoc.

Modulation of TARP γ8-Containing AMPA Receptors as a Novel Therapeutic Approach for Chronic Pain.

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.

Extracellular signal-regulated kinases mediate the enhancing effects of inflammatory mediators on resurgent currents in dorsal root ganglion neurons.

Previously we reported that a group of inflammatory mediators significantly enhanced resurgent currents in dorsal root ganglion neurons. To understand the underlying intracellular signaling mechanism, we investigated the effects of inhibition of extracellular signal-regulated kinases and protein kinase C on the enhancing effects of inflammatory mediators on resurgent currents in rat dorsal root ganglion neurons. We found that the extracellular signal-regulated kinases inhibitor U0126 completely prevented the enhancing effects of the inflammatory mediators on both Tetrodotoxin-sensitive and Tetrodotoxin-resistant resurgent currents in both small and medium dorsal root ganglion neurons. U0126 substantially reduced repetitive firing in small dorsal root ganglion neurons exposed to inflammatory mediators, consistent with prevention of resurgent current amplitude increases. The protein kinase C inhibitor Bisindolylmaleimide I also showed attenuating effects on resurgent currents, although to a lesser extent compared to extracellular signal-regulated kinases inhibition. These results indicate a critical role of extracellular signal-regulated kinases signaling in modulating resurgent currents and membrane excitability in dorsal root ganglion neurons treated with inflammatory mediators. It is also suggested that targeting extracellular signal-regulated kinases-resurgent currents might be a useful strategy to reduce inflammatory pain.

Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist.

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.

Broad spectrum efficacy with LY2969822, an oral prodrug of metabotropic glutamate 2/3 receptor agonist LY2934747, in rodent pain models.

BACKGROUND AND PURPOSE: A body of evidence suggests activation of metabotropic glutamate 2/3 (mGlu2/3 ) receptors would be an effective analgesic in chronic pain conditions. Thus, the analgesic properties of a novel mGlu2/3 receptor agonist prodrug were investigated. EXPERIMENTAL APPROACH: After oral absorption, the prodrug LY2969822 rapidly converts to the brain penetrant, potent and subtype-selective mGlu2/3 receptor agonist LY2934747. Behavioural assessments of allodynia, hyperalgesia and nocifensive behaviours were determined in preclinical pain models after administration of LY2969822 0.3-10 mg·kg-1 . In addition, the ability of i.v. LY2934747 to modulate dorsal horn spinal cord wide dynamic range (WDR) neurons in spinal nerve ligated (SNL) rats was assessed. KEY RESULTS: Following treatment with LY2934747, the spontaneous activity and electrically-evoked wind-up of WDR neurons in rats that had undergone spinal nerve ligation and developed mechanical allodynia were suppressed. In a model of sensitization, orally administered LY2969822 prevented the nociceptive behaviours induced by an intraplantar injection of formalin. The on-target nature of this effect was confirmed by blockade with an mGlu2/3 receptor antagonist. LY2969822 prevented capsaicin-induced tactile hypersensitivity, reversed the SNL-induced tactile hypersensitivity and reversed complete Freund's adjuvant - induced mechanical hyperalgesia. The mGlu2/3 receptor agonist prodrug demonstrated efficacy in visceral pain models, including a colorectal distension model and partially prevented the nocifensive behaviours in the mouse acetic acid writhing model. CONCLUSIONS AND IMPLICATIONS: Following oral administration of the prodrug LY2969822, the mGlu2/3 receptor agonist LY2934747 was formed and this attenuated pain behaviours across a broad range of preclinical pain models.

Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism.

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.

Protein kinase C enhances human sodium channel hNav1.7 resurgent currents via a serine residue in the domain III-IV linker.

Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7-mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation-mimicking mutations and were prevented by a phosphorylation-deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III-IV linker of sodium channels.

Tetrodotoxin-resistant sodium channels in sensory neurons generate slow resurgent currents that are enhanced by inflammatory mediators.

Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the ß4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel ß4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the ß4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.

A review of the abuse potential assessment of atomoxetine: a nonstimulant medication for attention-deficit/hyperactivity disorder.

RATIONALE: Treatment of attention-deficit/hyperactivity disorder (ADHD) has for many years relied on psychostimulants, particularly various formulations of amphetamines and methylphenidate. These are central nervous system stimulants and are scheduled because of their abuse potential. Atomoxetine (atomoxetine hydrochloride; Strattera®) was approved in 2002 for treatment of ADHD, and was the first nonstimulant medication approved for this disorder. It was classified as an unscheduled medication indicating a low potential for abuse. However, the abuse potential of atomoxetine has not been reviewed. OBJECTIVES: In this article, we review the evidence regarding abuse potential of atomoxetine, a selective inhibitor of the presynaptic norepinephrine transporter, which is unscheduled/unrestricted in all countries where it is approved. METHODS: Results from receptor binding, in vitro electrophysiology, in vivo microdialysis, preclinical behavioral, and human laboratory studies have been reviewed. RESULTS: Atomoxetine has no appreciable affinity for, or action at, central receptors through which drugs of abuse typically act, i.e., dopamine transporters, GABA(A) receptors, and opioid µ receptors. In behavioral experiments in rodents, atomoxetine does not increase locomotor activity, and in drug discrimination studies, its profile is similar to that of drugs without abuse potential. Atomoxetine does not serve as a reinforcer in monkey self-administration studies, and human laboratory studies suggest that atomoxetine does not induce subjective effects indicative of abuse. CONCLUSION: Neurochemical, preclinical, and early clinical studies predicted and supported a lack of abuse potential of atomoxetine, which is consistent with the clinical trial and postmarketing spontaneous event data in the past 10 years.

Glutamate receptor δ2 associates with metabotropic glutamate receptor 1 (mGluR1), protein kinase Cγ, and canonical transient receptor potential 3 and regulates mGluR1-mediated synaptic transmission in cerebellar Purkinje neurons.

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.

3-Phenyl-5-isothiazole carboxamides with potent mGluR1 antagonist activity.

The disclosed 3-phenyl-5-isothiazole carboxamides are potent allosteric antagonists of mGluR1 with generally good selectivity relative to the related group 1 receptor mGluR5. Pharmacokinetic properties of a member of this series (1R,2R)-N-(3-(4-methoxyphenyl)-4-methylisothiazol-5-yl)-2-methylcyclopropanecarboxamide (14) are good, showing acceptable plasma and brain exposure after oral dosing. Oral administration of isothiazole 14 gave robust activity in the formalin model of persistent pain which correlated with CNS receptor occupancy.

Structural and functional analysis of two new positive allosteric modulators of GluA2 desensitization and deactivation.

At the dimer interface of the extracellular ligand-binding domain of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors a hydrophilic pocket is formed that is known to interact with two classes of positive allosteric modulators, represented by cyclothiazide and the ampakine 2H,3H,6aH-pyrrolidino(2,1-3',2')1,3-oxazino(6',5'-5,4)benzo(e)1,4-dioxan-10-one (CX614). Here, we present structural and functional data on two new positive allosteric modulators of AMPA receptors, phenyl-1,4-bis-alkylsulfonamide (CMPDA) and phenyl-1,4-bis-carboxythiophene (CMPDB). Crystallographic data show that these compounds bind within the modulator-binding pocket and that substituents of each compound overlap with distinct moieties of cyclothiazide and CX614. The goals of the present study were to determine 1) the degree of modulation by CMPDA and CMPDB of AMPA receptor deactivation and desensitization; 2) whether these compounds are splice isoform-selective; and 3) whether predictions of mechanism of action could be inferred by comparing molecular interactions between the ligand-binding domain and each compound with those of cyclothiazide and CX614. CMPDB was found to be more isoform-selective than would be predicted from initial binding assays. It is noteworthy that these new compounds are both more potent and more effective and may be more clinically relevant than the AMPA receptor modulators described previously.

Evolving cancer pain treatments: rational approaches to improve the quality of life for cancer patients.

Most cancer patients will experience moderate to severe pain and/or neuropathy during the course of their disease. Recent improvements in the primary treatment of cancer have increased the life span of cancer patients, but not necessarily their quality of life (QoL). The pain and suffering cancer patients experience may be the result of the tumor itself, or the treatments required to arrest tumor growth and progression. In contrast to the rapid, highly mechanistic, tailored medicine approach used to target and treat the primary tumor burden, the evolution of pain and other supportive treatment approaches for cancer patients have been slow to non-existent. A movement is emerging to use more rational mechanistic approaches to the treatment of pain created by cancer and chemotherapeutics. This review briefly describes the most severe and debilitating symptoms (endophenotypes) from the cancer patient's perspective, the biochemical/neurobiological sequalae associated with tumor growth and therapies designed to arrest tumor progression, and highlights some promising pharmacologic mechanisms that may be used to treat cancer-related pain, sensory neuropathies, and associated endophenotypes. Delivering improved broader spectrum supportive care medicines to cancer patients will fill a significant unmet need and enable them to live productive, fulfilling lives that preserve their overall QoL.

Discovery of potent, cyclic calcitonin gene-related peptide receptor antagonists.

Calcitonin gene-related peptide (CGRP), a potent dilator of cerebral and dural vasculature, is known to be elevated in plasma and cerebral spinal fluid during migraine attacks. Selective blockade of the CGRP receptor offers the promise of controlling migraine headache more effectively and without the side-effects associated with the use of triptans. Our efforts to develop a novel, peptide-based CGRP antagonist focused on the C-terminal portion of the peptide which is known to bind the receptor but lack agonist properties. Extensive SAR studies of the C-terminal CGRP (27-37) region identified a novel cyclic structure: Bz-Val-Tyr-cyclo[Cys-Thr-Asp-Val-Gly-Pro-Phe-Cys]-Phe-NH(2) (23) with a kb value of 0.126 nM against the cloned human CGRP receptor. Additional SAR studies directed at enhancement of potency and improvement of physicochemical properties yielded a series of analogs with kb values in the 0.05-0.10 nM range.

Targeting group II metabotropic glutamate (mGlu) receptors for the treatment of psychosis associated with Alzheimer's disease: selective activation of mGlu2 receptors amplifies beta-amyloid toxicity in cultured neurons, whereas dual activation of mGlu2 and mGlu3 receptors is neuroprotective.

Dual orthosteric agonists of metabotropic glutamate 2 (mGlu2) and mGlu3 receptors are being developed as novel antipsychotic agents devoid of the adverse effects of conventional antipsychotics. Therefore, these drugs could be helpful for the treatment of psychotic symptoms associated with Alzheimer's disease (AD). In experimental animals, the antipsychotic activity of mGlu2/3 receptor agonists is largely mediated by the activation of mGlu2 receptors and is mimicked by selective positive allosteric modulators (PAMs) of mGlu2 receptors. We investigated the distinct influence of mGlu2 and mGlu3 receptors in mixed and pure neuronal cultures exposed to synthetic ß-amyloid protein (Aß) to model neurodegeneration occurring in AD. The mGlu2 receptor PAM, N-4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), devoid of toxicity per se, amplified Aß-induced neurodegeneration, and this effect was prevented by the mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). LY566332 potentiated Aß toxicity regardless of the presence of glial mGlu3 receptors, but it was inactive when neurons lacked mGlu2 receptors. The dual mGlu2/3 receptor agonist, (-)-2-oxa-4-aminobicyclo[3.1.0]exhane-4,6-dicarboxylic acid (LY379268), was neuroprotective in mixed cultures via a paracrine mechanism mediated by transforming growth factor-ß1. LY379268 lost its protective activity in neurons grown with astrocytes lacking mGlu3 receptors, indicating that protection against Aß neurotoxicity was mediated entirely by glial mGlu3 receptors. The selective noncompetitive mGlu3 receptor antagonist, (3S)-1-(5-bromopyrimidin-2-yl)-N-(2,4-dichlorobenzyl)pyrrolidin-3-amine methanesulfonate hydrate (LY2389575), amplified Aß toxicity on its own, and, interestingly, unmasked a neurotoxic activity of LY379268, which probably was mediated by the activation of mGlu2 receptors. These data indicate that selective potentiation of mGlu2 receptors enhances neuronal vulnerability to Aß, whereas dual activation of mGlu2 and mGlu3 receptors is protective against Aß-induced toxicity.

Hippocampal AMPA receptor gating controlled by both TARP and cornichon proteins.

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.

TARPs differentially decorate AMPA receptors to specify neuropharmacology.

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.

AMPA receptor subunit-specific regulation by a distinct family of type II TARPs.

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.

New transmembrane AMPA receptor regulatory protein isoform, gamma-7, differentially regulates AMPA receptors.

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.

Pharmacological and pharmacokinetic properties of a structurally novel, potent, and selective metabotropic glutamate 2/3 receptor agonist: in vitro characterization of agonist (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic acid (LY404039).

Group II metabotropic glutamate (mGlu) receptor agonists, including (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), have demonstrated efficacy in animal models of anxiety and schizophrenia, and LY354740 decreased anxiety in human subjects. Herein, we report the in vitro pharmacological profile and pharmacokinetic properties of another potent, selective, and structurally novel mGlu2/3 receptor agonist, (-)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) and provide comparisons with LY354740. Similar to LY354740, LY404039 is a nanomolar potent agonist at recombinant human mGlu2 and mGlu3 receptors (K(i) = 149 and 92, respectively) and in rat neurons expressing native mGlu2/3 receptors (Ki = 88). LY404039 is highly selective for mGlu2/3 receptors, showing more than 100-fold selectivity for these receptors, versus ionotropic glutamate receptors, glutamate transporters, and other receptors targeted by known anxiolytic and antipsychotic medications. Functionally, LY404039 potently inhibited forskolin-stimulated cAMP formation in cells expressing human mGlu2 and mGlu3 receptors. Electrophysiological studies indicated that LY404039 suppressed electrically evoked excitatory activity in the striatum, and serotonin-induced l-glutamate release in the prefrontal cortex; effects reversed by LY341495. These characteristics suggest LY404039 modulates glutamatergic activity in limbic and forebrain areas relevant to psychiatric disorders; and that, similar to LY354740, it works through a mechanism that may be devoid of negative side effects associated with current antipsychotics and anxiolytics. Interestingly, despite the slightly lower potency (approximately 2-5-fold) of LY404039 versus LY354740 in binding, functional, and electrophysiological assays, LY404039 demonstrated higher plasma exposure and better oral bioavailability in pharmacokinetic experiments. Collectively, the current data indicate that LY404039 may be valuable in the treatment of neuropsychiatric disorders, including anxiety and psychosis.