Stephen J. Moss

GABA A -receptor-associated protein links GABA A receptors and the cytoskeleton

Type-A receptors for the neurotransmitter GABA (γ-aminobutyric acid) are ligand-gated chloride channels that mediate inhibitory neurotransmission. Each subunit of the pentameric receptor protein has ligand-binding sites in the amino-terminal extracellular domain and four membrane-spanning regions, one of which forms a wall of the ion channel. Each subunit also has a large intracellular loop that may be a target for protein kinases and be required for subcellular targeting and membrane clustering of the receptor, perhaps by anchoring the receptor to the cytoskeleton. Neurotransmitter receptors need to be positioned in high density in the cell membrane at sites postsynaptic to nerve terminals releasing that neurotransmitter. Other members of the superfamily of ligand-gated ion-channel receptors associate in postsynaptic-membrane clusters by binding to the proteins rapsyn or gephyrin. Here we identify a new cellular protein, GABAA-receptor-associated protein (GABARAP), which can interact with the γ2 subunit of GABAA receptors. GABARAP binds to GABAA receptors both in vitro and in vivo, and co-localizes with the punctate staining of GABAA receptors on cultured cortical neurons. Sequence analysis shows similarity between GABARAP and light chain-3 of microtubule-associated proteins 1A and 1B. Moreover, the N terminus of GABARAP is highly positively charged and features a putative tubulin-binding motif. The interactions among GABAA receptors, GABARAP and tubulin suggest a mechanism for the targeting and clustering of GABAA receptors.

Antibodies to the GABAB receptor in limbic encephalitis with seizures: case series and characterisation of the antigen

BACKGROUND Some encephalitides or seizure disorders once thought idiopathic now seem to be immune mediated. We aimed to describe the clinical features of one such disorder and to identify the autoantigen involved. METHODS 15 patients who were suspected to have paraneoplastic or immune-mediated limbic encephalitis were clinically assessed. Confocal microscopy, immunoprecipitation, and mass spectrometry were used to characterise the autoantigen. An assay of HEK293 cells transfected with rodent GABA(B1) or GABA(B2) receptor subunits was used as a serological test. 91 patients with encephalitis suspected to be paraneoplastic or immune mediated and 13 individuals with syndromes associated with antibodies to glutamic acid decarboxylase 65 were used as controls. FINDINGS All patients presented with early or prominent seizures; other symptoms, MRI, and electroencephalography findings were consistent with predominant limbic dysfunction. All patients had antibodies (mainly IgG1) against a neuronal cell-surface antigen; in three patients antibodies were detected only in CSF. Immunoprecipitation and mass spectrometry showed that the antibodies recognise the B1 subunit of the GABA(B) receptor, an inhibitory receptor that has been associated with seizures and memory dysfunction when disrupted. Confocal microscopy showed colocalisation of the antibody with GABA(B) receptors. Seven of 15 patients had tumours, five of which were small-cell lung cancer, and seven patients had non-neuronal autoantibodies. Although nine of ten patients who received immunotherapy and cancer treatment (when a tumour was found) showed neurological improvement, none of the four patients who were not similarly treated improved (p=0.005). Low levels of GABA(B1) receptor antibodies were identified in two of 104 controls (p<0.0001). INTERPRETATION GABA(B) receptor autoimmune encephalitis is a potentially treatable disorder characterised by seizures and, in some patients, associated with small-cell lung cancer and with other autoantibodies. FUNDING National Institutes of Health.

Constructing inhibitory synapses

Control of nerve-cell excitability is crucial for normal brain function. Two main groups of inhibitory neurotransmitter receptors — GABAA and glycine receptors — fulfil a significant part of this role. To mediate fast synaptic inhibition effectively, these receptors need to be localized and affixed opposite nerve terminals that release the appropriate neurotransmitter at multiple sites on postsynaptic neurons. But for this to occur, neurons require intracellular anchoring molecules, as well as mechanisms that ensure the efficient turnover and transport of mature, functional inhibitory synaptic receptor proteins. This review describes the dynamic regulation of synaptic GABAA and glycine receptors and discusses recent advances in this rapidly evolving field.

GABAA receptor trafficking and its role in the dynamic modulation of neuronal inhibition

GABA (γ-aminobutyric acid) type A receptors (GABAARs) mediate most fast synaptic inhibition in the mammalian brain, controlling activity at both the network and the cellular levels. The diverse functions of GABA in the CNS are matched not just by the heterogeneity of GABAARs, but also by the complex trafficking mechanisms and protein–protein interactions that generate and maintain an appropriate receptor cell-surface localization. In this Review, we discuss recent progress in our understanding of the dynamic regulation of GABAAR composition, trafficking to and from the neuronal surface, and lateral movement of receptors between synaptic and extrasynaptic locations. Finally, we highlight a number of neurological disorders, including epilepsy and schizophrenia, in which alterations in GABAAR trafficking occur.

Activation of estrogen receptor-beta regulates hippocampal synaptic plasticity and improves memory.

Estrogens have long been implicated in influencing cognitive processes, yet the molecular mechanisms underlying these effects and the roles of the estrogen receptors alpha (ERα) and beta (ERβ) remain unclear. Using pharmacological, biochemical and behavioral techniques, we demonstrate that the effects of estrogen on hippocampal synaptic plasticity and memory are mediated through ERβ. Selective ERβ agonists increased key synaptic proteins in vivo, including PSD-95, synaptophysin and the AMPA-receptor subunit GluR1. These effects were absent in ERβ knockout mice. In hippocampal slices, ERβ activation enhanced long-term potentiation, an effect that was absent in slices from ERβ knockout mice. ERβ activation induced morphological changes in hippocampal neurons in vivo, including increased dendritic branching and increased density of mushroom-type spines. An ERβ agonist, but not an ERα agonist, also improved performance in hippocampus-dependent memory tasks. Our data suggest that activation of ERβ can regulate hippocampal synaptic plasticity and improve hippocampus-dependent cognition.

Regulation of GABAA receptor function by protein kinase C phosphorylation.

GABAA receptors possess consensus sequences for phosphorylation by PKC that are located on the presumed intracellular domains of beta and gamma 2 subunits. PKC phosphorylation sites were analyzed using purified receptor subunits and were located on up to 3 serine residues in beta 1 and gamma 2 subunits. The role of phosphorylation in receptor function was studied using recombinant receptors expressed in kidney cells and Xenopus oocytes and was compared with native neuronal GABAA receptors. For recombinant and native GABAA receptors, PKC phosphorylation caused a reduction in the amplitudes of GABA-activated currents without affecting the time constants for current decay. Selective site-directed mutagenesis of the serine residues reduced the effects of phorbol esters and revealed that serine 343 in the gamma 2 subunit exerted the largest effect on the GABA-activated response. These results indicate that PKC phosphorylation can differentially modulate GABAA receptor function.

Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition.

Fast synaptic inhibition in the brain is largely mediated by GABA(A) receptors. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. It has become clear that the stability and activity of GABA(A) receptors at synapses can be dynamically modulated by receptor trafficking and phosphorylation. Here, we discuss these regulatory mechanisms, and their consequences for the efficacy of GABA(A) receptor mediated synaptic inhibition.

Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, activity, and cell-surface stability.

The efficacy of GABAergic synaptic inhibition is a principal factor in controlling neuronal activity. We demonstrate here that brain-derived neurotrophic factor modulates the activity of GABAA receptors, the main sites of fast synaptic inhibition in the brain, within minutes of application. Temporally, this comprised an early enhancement in the miniature IPSC amplitude, followed by a prolonged depression. This modulation was concurrent with enhanced PKC-mediated phosphorylation, followed by protein phosphatase 2A (PP2A)-mediated dephosphorylation of the GABAA receptor. Mechanistically, these events were facilitated by differential recruitment of PKC, receptor for activated C-kinase, and PP2A to GABAA receptors, depending on the phosphorylation state of the receptor β3-subunit. Thus, transient formation of GABAA receptor signaling complexes has the potential to provide a basis for acute changes in receptor function underlying GABAergic synaptic plasticity.

Biochemical Characterization and Localization of a Non-N-Methyl-D-Aspartate Glutamate Receptor in Rat Brain

Abstract: The structure and distribution of non‐N‐methyl‐D‐aspartate glutamate receptors in the rat brain were studied using subunit‐specific antibodies that recognize the receptor subunit GluRl. The GluRl protein, a 106‐kDa glycoprotein, appears predominantly in synaptic plasma membranes, where it is highly enriched in the postsynaptic densities. When synaptic plasma membranes are solubilized with the detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐l‐propanesul‐fonate, high‐affinity a‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) binding and GluRl immunoreactivity comigrate at a native Mr of 610,000. GluRl is enriched in the hippocampus and cerebellar cortex but is present throughout the CNS. It is found on neuronal cell bodies and processes within most regions of the brain; within the cerebellum, however, it is localized to the Bergmann glia. These data suggest that the GluRl protein is a subunit of multimeric AMPA‐preferring glutamate receptors present on neurons and on specialized glia.

GABA A receptor cell surface number and subunit stability are regulated by the ubiquitin-like protein Plic-1

Controlling the number of functional γ-aminobutyric acid A (GABAA) receptors in neuronal membranes is a crucial factor for the efficacy of inhibitory neurotransmission. Here we describe the direct interaction of GABAA receptors with the ubiquitin-like protein Plic-1. Furthermore, Plic-1 is enriched at inhibitory synapses and is associated with subsynaptic membranes. Functionally, Plic-1 facilitates GABAA receptor cell surface expression without affecting the rate of receptor internalization. Plic-1 also enhances the stability of intracellular GABAA receptor subunits, increasing the number of receptors available for insertion into the plasma membrane. Our study identifies a previously unknown role for Plic-1, a modulation of GABAA receptor cell surface number, which suggests that Plic-1 facilitates accumulation of these receptors in dendritic membranes.