Sustained Inhibition of GABA-AT by OV329 Enhances Neuronal Inhibition and Prevents Development of Benzodiazepine Refractory Seizures.

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain which mediates its rapid effects on neuronal excitability via ionotropic GABA receptors. GABA levels in the brain are critically dependent upon GABA-aminotransferase (GABA-AT) which promotes its degradation. Vigabatrin, a low-affinity GABA-AT inhibitor, exhibits anticonvulsant efficacy, but its use is limited due to cumulative ocular toxicity.

The brain-specific kinase LMTK3 regulates neuronal excitability by decreasing KCC2-dependent neuronal Cl extrusion.

LMTK3 is a brain-specific transmembrane serine/threonine protein kinase that acts as a scaffold for protein phosphatase-1 (PP1). Although LMKT3 has been identified as a risk factor for autism and epilepsy, its physiological significance is unknown. Here, we demonstrate that LMTK3 copurifies and binds to KCC2, a neuron-specific K/Cl transporter.

Spectrin-beta 2 facilitates the selective accumulation of GABA receptors at somatodendritic synapses.

Fast synaptic inhibition is dependent on targeting specific GABAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABARs are typically assembled from α1-3, β and γ subunits. Here, we isolate distinct GABARs from the brain and interrogate their composition using quantitative proteomics.

Analyzing the mechanisms that facilitate the subtype-specific assembly of -aminobutyric acid type A receptors.

Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic -aminobutyric acid type A receptors (GABA Rs), which mediate phasic and tonic inhibition, respectively. These two GABA R subtypes differ in their function, ligand sensitivity, and physiological properties.

Preventing Phosphorylation of the GABA R β3 Subunit Compromises the Behavioral Effects of Neuroactive Steroids.

Neuroactive steroids (NASs) have potent anxiolytic, anticonvulsant, sedative, and hypnotic actions, that reflect in part their efficacy as GABA R positive allosteric modulators (PAM). In addition to this, NAS exert metabotropic effects on GABAergic inhibition the activation of membrane progesterone receptors (mPRs), which are G-protein coupled receptors. mPR activation enhances the phosphorylation of residues serine 408 and 409 (S408/9) in the β3 subunit of GABA Rs, increasing their accumulation in the plasma membrane leading to a sustained increase in tonic inhibition.

Discovery and functional characterization of N-(thiazol-2-yl)-benzamide analogs as the first class of selective antagonists of the Zinc-Activated Channel (ZAC).

The Zinc-Activated Channel (ZAC) is an atypical member of the Cys-loop receptor (CLR) superfamily of pentameric ligand-gated ion channels, with its very different endogenous agonists and signalling properties. In this study, a compound library screening at ZAC resulted in the identification of 2-(5-bromo-2-chlorobenzamido)-4-methylthiazole-5-methyl ester (1) as a novel ZAC antagonist. The structural determinants for ZAC activity in 1 were investigated by functional characterization of 61 analogs at ZAC expressed in Xenopus oocytes by two-electrode voltage clamp electrophysiology, and couple of analogs exerting more potent ZAC inhibition than 1 were identified (IC values: 1-3 μM).

Probing the molecular basis for signal transduction through the Zinc-Activated Channel (ZAC).

The molecular basis for the signal transduction through the classical Cys-loop receptors (CLRs) has been delineated in great detail. The Zinc-Activated Channel (ZAC) constitutes a so far poorly elucidated fifth branch of the CLR superfamily, and in this study we explore the molecular mechanisms underlying ZAC signaling in Xenopus oocytes by two-electrode voltage clamp electrophysiology. In studies of chimeric receptors fusing either the extracellular domain (ECD) or the transmembrane/intracellular domain (TMD-ICD) of ZAC with the complementary domains of 5-HTA serotonin or α glycine receptors, serotonin and Zn/H evoked robust concentration-dependent currents in 5-HTA/ZAC- and ZAC/α-Gly-expressing oocytes, respectively, suggesting that Zn and protons activate ZAC predominantly through its ECD.

Delineation of the functional properties exhibited by the Zinc-Activated Channel (ZAC) and its high-frequency ThrAla variant (rs2257020) in Xenopus oocytes.

The signalling characteristics of the Zinc-Activated Channel (ZAC), a member of the Cys-loop receptor (CLR) superfamily, are presently poorly elucidated. The ZACN polymorphism c.454G>A encoding for the ThrAla variation in ZAC is found in extremely high allele frequencies across ethnicities.

KCC2 is required for the survival of mature neurons but not for their development.

The K/Cl cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity.

Isolation and Characterization of Multi-Protein Complexes Enriched in the K-Cl Co-transporter 2 From Brain Plasma Membranes.

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification.

Inhibitory Synapse Formation at the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABARs).

Identification of a Core Amino Acid Motif within the α Subunit of GABARs that Promotes Inhibitory Synaptogenesis and Resilience to Seizures.

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABARs) at the appropriate synaptic sites. Synaptic GABARs are constructed from α(1-3), β(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABARs and the inhibitory scaffold proteins collybistin and gephyrin.

Metabotropic, but not allosteric, effects of neurosteroids on GABAergic inhibition depend on the phosphorylation of GABA receptors.

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs).

Neuroactive Steroids Reverse Tonic Inhibitory Deficits in Fragile X Syndrome Mouse Model.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. A reduction in neuronal inhibition mediated by γ-aminobutyric acid type A receptors (GABARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAR channel function, but recent studies from our laboratory have revealed that NASs also exert persistent metabotropic effects on the efficacy of tonic inhibition by increasing the protein kinase C (PKC)-mediated phosphorylation of the α4 and β3 subunits which increase the membrane expression and boosts tonic inhibition.

Endogenous and synthetic neuroactive steroids evoke sustained increases in the efficacy of GABAergic inhibition via a protein kinase C-dependent mechanism.

The neuroactive steroid (NAS) tetrahydrodeoxycorticosterone (THDOC) increases protein kinase C (PKC) mediated phosphorylation of extrasynaptic GABA receptor (GABAR) subunits leading to increased surface expression of α4/β3 subunit-containing extrasynaptic GABARs, leading to a sustained increase in GABAR tonic current density. Whether other naturally occurring and synthetic NASs share both an allosteric and metabotropic action on GABARs is unknown. Here, we examine the allosteric and metabotropic properties of allopregnanolone (ALLO), and synthetic NASs SGE-516 and ganaxolone.

Regulating the Efficacy of Inhibition Through Trafficking of γ-Aminobutyric Acid Type A Receptors.

Trafficking of anesthetic-sensitive receptors within the plasma membrane, or from one cellular component to another, occurs continuously. Changes in receptor trafficking have implications in altering anesthetic sensitivity. γ-Aminobutyric acid type A receptors (GABAARs) are anion-permeable ion channels and are the major class of receptor in the adult mammalian central nervous system that mediates inhibition.

Compromising KCC2 transporter activity enhances the development of continuous seizure activity.

Impaired neuronal inhibition has long been associated with the increased probability of seizure occurrence and heightened seizure severity. Fast synaptic inhibition in the brain is primarily mediated by the type A γ-aminobutyric acid receptors (GABAARs), ligand-gated ion channels that can mediate Cl(-) influx resulting in membrane hyperpolarization and the restriction of neuronal firing. In most adult brain neurons, the K(+)/Cl(-) co-transporter-2 (KCC2) establishes hyperpolarizing GABAergic inhibition by maintaining low [Cl(-)]i.

Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse.

The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, β, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2).

Copper and protons directly activate the zinc-activated channel.

The zinc-activated channel (ZAC) is a cationic ion channel belonging to the superfamily of Cys-loop receptors, which consists of pentameric ligand-gated ion channels. ZAC is the least understood member of this family so in the present study we sought to characterize the properties of this channel further. We demonstrate that not only zinc (Zn(2+)) but also copper (Cu(2+)) and protons (H(+)) are agonists of ZAC, displaying potencies and efficacies in the rank orders of H(+)>Cu(2+)>Zn(2+) and H(+)>Zn(2+)>Cu(2+), respectively.

Compromising the phosphodependent regulation of the GABAAR β3 subunit reproduces the core phenotypes of autism spectrum disorders.

Alterations in the efficacy of neuronal inhibition mediated by GABAA receptors (GABAARs) containing β3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABAARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the β3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in β3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A).

KCC2 activity is critical in limiting the onset and severity of status epilepticus.

The K(+)/Cl(-) cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl(-) levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940).

Phosphorylation of GABAA receptors influences receptor trafficking and neurosteroid actions.

Rationale: Gamma-aminobutyric acid type A receptors (GABAARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABAARs can be localized at post-synaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABAARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABAARs are typically activated continuously by ambient GABA concentrations and thus mediate tonic inhibition.

Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors.

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions.