Martin B. Gill

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.

Hippocampal NMDA receptor subunits differentially regulate fear memory formation and neuronal signal propagation.

Activation of NMDA receptors (NMDAR) in the hippocampus is essential for the formation of contextual and trace memory. However, the role of individual NMDAR subunits in the molecular mechanisms contributing to these memory processes is not known. Here we demonstrate, using intrahippocampal injection of subunit‐selective compounds, that the NR2A‐preferring antagonist impaired contextual and trace fear conditioning as well as learning‐induced increase of the nuclear protein c‐Fos. The NR2B‐specific antagonist, on the other hand, selectively blocked trace fear conditioning without affecting c‐Fos levels. Studies with cultured primary hippocampal neurons, further showed that synaptic and extrasynaptic NR2A and NR2B differentially regulate the extracellular signal‐regulated kinase 1 and 2/mitogen‐ and stress‐activated protein kinase 1 (ERK1/2/MSK1)/c‐Fos pathway. Activation of the synaptic population of NMDAR induced cytosolic, cytoskeletal, and perinuclear phosphorylation of ERK1/2 (pERK1/2). The nuclear propagation of pERK1/2 signals, revealed by upregulation of the downstream nuclear targets pMSK1 and c‐Fos, was blocked by a preferential NR2A but not by a specific NR2B antagonist. Conversely, activation of total (synaptic and extrasynaptic) NMDAR engaged receptors with NR2B subunits, and resulted in membrane retention of pERK1/2 without inducing pMSK1 and c‐Fos. Stimulation of extrasynaptic NMDAR alone was consistently ineffective at activating ERK signaling. The discrete contribution of synaptic and total NR2A‐ and NR2B‐containing NMDAR to nuclear transmission vs. membrane retention of ERK signaling may underlie their specific roles in the formation of contextual and trace fear memory.

Cornichon-2 Modulates AMPA Receptor–Transmembrane AMPA Receptor Regulatory Protein Assembly to Dictate Gating and Pharmacology

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.

Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an “open” conformation, or “lock” the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.

Novel N-Methylated 8-Oxoisoguanines from Pacific Sponges with Diverse Neuroactivities

Marine organisms have yielded a variety of metabolites with neuropharmacological applications. Here we describe the isolation and pharmacological characterization of four novel, neurologically active purines 1-4, isolated from Haplosclerida sponges collected in the Republic of Palau. The structures were determined by analyses of spectral and X-ray data. Compound 1 induced convulsions upon intracerebroventricular injection into mice, with a CD50 value of 2.4 nmol/mouse. Purines 2-4 were active in mouse bioassays at higher doses. The seizurogenic activity of 1 was correlated with inhibition of neuronal GABAergic transmission, with only a modest impact on excitatory signaling, in electrophysiological recordings from hippocampal neurons. Despite having a purine template structure, the inhibitory activity of 1 was not prevented by a nonselective adenosine receptor antagonist. Thus, 1 represents a novel substituted purine that elicits convulsions through its actions on inhibitory neurotransmission. These 8-oxoisoguanine analogs comprise a new family of compounds closely related in structure to endogenous neurosignaling molecules and commonly used CNS stimulants.

AMPA receptor modulation by cornichon‐2 dictated by transmembrane AMPA receptor regulatory protein isoform

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 IKA/IGlu 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.

Transmembrane AMPA receptor regulatory proteins and cornichon-2 allosterically regulate AMPA receptor antagonists and potentiators

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 [3H]-LY450295 and confers sensitivity to displacement by non-competitive antagonists. In cerebellar membranes from stargazer mice, [3H]-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 [3H]-LY450295 potentiator binding to displacement by non-competitive antagonists. Autoradiography of [3H]-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.

A series of structurally novel heterotricyclic α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor-selective antagonists

Background and purpose:  A new class of heterotricyclic glutamate analogues recently was generated by incorporating structural elements of two excitotoxic marine compounds, kainic acid and neodysiherbaine A. Rather than acting as convulsants, several of these ‘IKM’ compounds markedly depressed CNS activity in mice. Here, we characterize the pharmacological profile of the series with a focus on the most potent of these molecules, IKM‐159.

An Emerging Role for TARPs in Neuropsychiatric Disorders

The discovery of glutamate as the principal excitatory neurotransmitter in brain was followed by the identification and molecular cloning of the ionotropic glutamate receptor family, which comprises NMDA, AMPA, and kainate receptors. The AMPA receptor subfamily mediates fast synaptic neurotransmission important to diverse sensory, behavioral, and cognitive processes, including learning and memory. However, excessive AMPA receptor activity and subsequent excitotoxicity underlie central nervous system disorders ranging from stroke to epilepsy. It was hypothesized that the development of AMPA receptor antagonists to dampen aberrant neurotransmission would provide treatments for neurological illnesses. However, clinical trials have shown side effects for these antagonists and suggested a need for greater selectivity. A seminal finding in AMPA receptor biology and neurotransmission was the discovery of stargazin, the protein mutated in stargazer mice, which show absence epilepsy and cerebellar ataxia. Cell biological and physiological studies showed that stargazin is an AMPA receptor auxiliary subunit and controls receptor trafficking, gating, and pharmacology. Subsequent studies have identified a family of related transmembrane AMPA receptor regulator proteins (TARPs). TARPs comprise γ-2 (stargazin), -3, -4, -5, -7, and -8 subunits, which are discretely distributed in specific neuronal and glial populations throughout the brain. Studies involving γ-8 knockout mice, which exhibit deficiencies in hippocampal neurotransmission, underscore the importance of TARPs both in region-specific control of AMPA receptor signaling, and in neurological disease—hippocampal excitotoxicity elicited by the AMPA receptor partial agonist, kainate, is abrogated in γ-8 knockout mice (Tomita et al, 2007). Neuropsychiatric conditions such as schizophrenia, depression, and bipolar disorder are severe, multifactorial brain illnesses of mood, cognition, and behavior whose etiologies remain uncertain. Molecular analyses have found abnormal expression for key components of glutamatergic neurotransmission, including TARPs. Increased and decreased stargazin mRNA expression has been documented in post-mortem schizophrenic and major depressive disorder brains, respectively (Beneyto and Meador-Woodruff, 2006). Silberberg et al (2008) found certain CACNG2 (the gene that encodes stargazin) allelic polymorphisms are associated with improved response to lithium, the classical treatment for bipolar disorder. Furthermore, chronic treatment with the antidepressants desipramine and paroxetine increased AMPA receptor association with stargazin in rat hippocampus (Martinez-Turrillas et al, 2007). Patients with bipolar disorder or schizophrenia have exhibited decreased CACNG2 DNA copy number (Wilson et al, 2006). Yet, increased stargazin mRNA expression has been found in the dorsolateral prefrontal cortex of brains from bipolar disorder patients suggesting a potential regio-specific action for stargazin in this disorder (Silberberg et al, 2008). Furthermore, the PDE 11A knockout mouse, which shows multiple psychiatric illness-related phenotypes, possessed decreased hippocampal expression of both γ-2 and -8 proteins (Kelly et al, 2010). Recent research into neuropsychiatric illnesses has shown an emerging pathological role for TARPs. As TARPs are differentially localized in neuron pathways, targeting individual isoforms may enable selective modulation of specific brain circuits without globally affecting synaptic transmission. However, the feasibility of uniquely targeting specific AMPA receptor complexes has not yet been established.

Isolation, amino acid sequence and biological activities of novel long-chain polyamine-associated peptide toxins from the sponge Axinyssa aculeata.

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N‐terminal residues that are modified by the addition of long‐chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU‐A and B, which were purified in this study, share a common polypeptide chain but differ in their N‐terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU‐A and B was deduced from 3′ and 5′ RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.