Mechanisms of inhibition and activation of extrasynaptic αß GABAA receptors.

Type A GABA (γ-aminobutyric acid) receptors represent a diverse population in the mammalian brain, forming pentamers from combinations of α-, ß-, γ-, δ-, ε-, ρ-, θ- and π-subunits1. αß, α4ßδ, α6ßδ and α5ßγ receptors favour extrasynaptic localization, and mediate an essential persistent (tonic) inhibitory conductance in many regions of the mammalian brain1,2. Mutations of these receptors in humans are linked to epilepsy and insomnia3,4. Altered extrasynaptic receptor function is implicated in insomnia, stroke and Angelman and Fragile X syndromes1,5, and drugs targeting these receptors are used to treat postpartum depression6. Tonic GABAergic responses are moderated to avoid excessive suppression of neuronal communication, and can exhibit high sensitivity to Zn2+ blockade, in contrast to synapse-preferring α1ßγ, α2ßγ and α3ßγ receptor responses5,7-12. Here, to resolve these distinctive features, we determined structures of the predominantly extrasynaptic αß GABAA receptor class. An inhibited state bound by both the lethal paralysing agent α-cobratoxin13 and Zn2+ was used in comparisons with GABA-Zn2+ and GABA-bound structures. Zn2+ nullifies the GABA response by non-competitively plugging the extracellular end of the pore to block chloride conductance. In the absence of Zn2+, the GABA signalling response initially follows the canonical route until it reaches the pore. In contrast to synaptic GABAA receptors, expansion of the midway pore activation gate is limited and it remains closed, reflecting the intrinsic low efficacy that characterizes the extrasynaptic receptor. Overall, this study explains distinct traits adopted by αß receptors that adapt them to a role in tonic signalling.

Presynaptic hyperexcitability reversed by positive allosteric modulation of a GABABR epilepsy variant.

GABABRs are key membrane proteins that continually adapt the excitability of the nervous system. These G-protein coupled receptors are activated by the brain's premier inhibitory neurotransmitter GABA. They are obligate heterodimers composed of GABA-binding GABABR1 and G-protein-coupling GABABR2 subunits. Recently, three variants (G693W, S695I, I705N) have been identified in the gene (GABBR2) encoding for GABABR2. Individuals that harbour any of these variants exhibit severe developmental epileptic encephalopathy and intellectual disability, but the underlying pathogenesis that is triggered in neurons, remains unresolved. Using a range of confocal imaging, flow cytometry, structural modelling, biochemistry, live cell Ca2+ imaging of presynaptic terminals, whole-cell electrophysiology of HEK-293T cells and neurons, and two-electrode voltage clamping of Xenopus oocytes we have probed the biophysical and molecular trafficking and functional profiles of G693W, S695I and I705N variants. We report that all three point mutations impair neuronal cell surface expression of GABABRs, reducing signalling efficacy. However, a negative effect evident for one variant perturbed neurotransmission by elevating presynaptic Ca2+ signalling. This is reversed by enhancing GABABR signalling via positive allosteric modulation. Our results highlight the importance of studying neuronal receptors expressed in nervous system tissue and provide new mechanistic insights into how GABABR variants can initiate neurodevelopmental disease whilst highlighting the translational suitability and therapeutic potential of allosteric modulation for correcting these deficits.

Differential Coassembly of α1-GABAARs Associated with Epileptic Encephalopathy.

GABAA receptors (GABAARs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren syndrome identified a frameshifting de novo variant in a major GABAAR gene, GABRA1 This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25 Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with ß3, but not with ß2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential ß subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition.SIGNIFICANCE STATEMENT GABAARs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain, and their dysfunction underlies many neurologic disorders, especially epilepsy. Here we report the characterization of an α1-GABAAR variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1 subunit cell surface expression with ß subunits resulting in severely reduced synaptic inhibition. Our study reveals that disease-inducing variants can affect GABAAR structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.

Cell surface expression of homomeric GABAA receptors depends on single residues in subunit transmembrane domains.

Cell surface expression of type A GABA receptors (GABAARs) is a critical determinant of the efficacy of inhibitory neurotransmission. Pentameric GABAARs are assembled from a large pool of subunits according to precise co-assembly rules that limit the extent of receptor structural diversity. These rules ensure that particular subunits, such as ρ1 and ß3, form functional cell surface ion channels when expressed alone in heterologous systems, whereas other brain-abundant subunits, such as α and γ, are retained within intracellular compartments. Why some of the most abundant GABAAR subunits fail to form homomeric ion channels is unknown. Normally, surface expression of α and γ subunits requires co-assembly with ß subunits via interactions between their N-terminal sequences in the endoplasmic reticulum. Here, using molecular biology, imaging, and electrophysiology with GABAAR chimeras, we have identified two critical residues in the transmembrane domains of α and γ subunits, which, when substituted for their ρ1 counterparts, permit cell surface expression as homomers. Consistent with this, substitution of the ρ1 transmembrane residues for the α subunit equivalents reduced surface expression and altered channel gating, highlighting their importance for GABAAR trafficking and signaling. Although not ligand-gated, the formation of α and γ homomeric ion channels at the cell surface was revealed by incorporating a mutation that imparts the functional signature of spontaneous channel activity. Our study identifies two single transmembrane residues that enable homomeric GABAAR subunit cell surface trafficking and demonstrates that α and γ subunits can form functional ion channels.

Context-Dependent Modulation of GABAAR-Mediated Tonic Currents.

Tonic GABA currents mediated by high-affinity extrasynaptic GABAA receptors, are increasingly recognized as important regulators of cell and neuronal network excitability. Dysfunctional GABAA receptor signaling that results in modified tonic GABA currents is associated with a number of neurological disorders. Consequently, developing compounds to selectively modulate the activity of extrasynaptic GABAA receptors underlying tonic inhibition is likely to prove therapeutically useful. Here, we examine the GABAA receptor subtype selectivity of the weak partial agonist, 5-(4-piperidyl)isoxazol-3-ol (4-PIOL), as a potential mechanism for modulating extrasynaptic GABAA receptor-mediated tonic currents. By using recombinant GABAA receptors expressed in HEK293 cells, and native GABAA receptors of cerebellar granule cells, hippocampal neurons, and thalamic relay neurons, 4-PIOL evidently displayed differential agonist and antagonist-type profiles, depending on the extrasynaptic GABAA receptor isoforms targeted. For neurons, this resulted in differential modulation of GABA tonic currents, depending on the cell type studied, their respective GABAA receptor subunit compositions, and critically, on the ambient GABA levels. Unexpectedly, 4-PIOL revealed a significant population of relatively low-affinity γ2 subunit-containing GABAA receptors in the thalamus, which can contribute to tonic inhibition under specific conditions when GABA levels are raised. Together, these data indicate that partial agonists, such as 4-PIOL, may be useful for modulating GABAA receptor-mediated tonic currents, but the direction and extent of this modulation is strongly dependent on relative expression levels of different extrasynaptic GABAA receptor subtypes, and on the ambient GABA levels. SIGNIFICANCE STATEMENT: A background level of inhibition (tonic) is important in the brain for controlling neuronal excitability. Increased levels of tonic inhibition are associated with some neurological disorders but there are no specific ligands capable of selectively reducing tonic inhibition. Here we explore the use of a GABA partial agonist as a selective chemical tool in three different brain regions. We discover that the activity of a partial agonist is heavily dependent upon the GABAA receptor subunit composition underpinning tonic inhibition, and on the ambient levels of GABA in the brain.

Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation.

KEY POINTS: Hyperekplexia or startle disease is a serious neurological condition affecting newborn children and usually involves dysfunctional glycinergic neurotransmission. Glycine receptors (GlyRs) are major mediators of inhibition in the spinal cord and brainstem. A missense mutation, replacing asparagine (N) with lysine (K), at position 46 in the GlyR α1 subunit induced hyperekplexia following a reduction in the potency of the transmitter glycine; this resulted from a rapid deactivation of the agonist current at mutant GlyRs. These effects of N46K were rescued by mutating a juxtaposed residue, N61 on binding Loop D, suggesting these two asparagines may interact. Asparagine 46 is considered to be important for the structural stability of the subunit interface and glycine binding site, and its mutation represents a new mechanism by which GlyR dysfunction induces startle disease. ABSTRACT: Dysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, ß-alanine and taurine by 9-, 6- and 3-fold respectively, and that of the competitive antagonist strychnine by 15-fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co-mutating N61, located on a neighbouring ß loop to N46, rescued the wild-type phenotype depending on the amino acid charge. Single-channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Brief report: isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration.

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.

Effects of Gabra2 Point Mutations on Alcohol Intake: Increased Binge-Like and Blunted Chronic Drinking by Mice.

BACKGROUND: Alcohol use disorders are associated with single-nucleotide polymorphisms in GABRA2, the gene encoding the GABAA receptor α2-subunit in humans. Deficient GABAergic functioning is linked to impulse control disorders, intermittent explosive disorder, and to drug abuse and dependence, yet it remains unclear whether α2-containing GABAA receptor sensitivity to endogenous ligands is involved in excessive alcohol drinking. METHODS: Male wild-type (Wt) C57BL/6J and point-mutated mice rendered insensitive to GABAergic modulation by benzodiazepines (BZD; H101R), allopregnanolone (ALLO) or tetrahydrodeoxycorticosterone (THDOC; Q241M), or high concentrations of ethanol (EtOH) (S270H/L277A) at α2-containing GABAA receptors were assessed for their binge-like, moderate, or escalated chronic drinking using drinking in the dark, continuous access (CA) and intermittent access (IA) to alcohol protocols, respectively. Social approach by mutant and Wt mice in forced alcohol abstinence was compared to approach by EtOH-naïve controls. Social deficits in forced abstinence were treated with allopregnanolone (0, 3.0, 10.0 mg/kg, intraperitoneal [i.p.]) or midazolam (0, 0.56, 1.0 mg/kg, i.p.). RESULTS: Mice with BZD-insensitive α2-containing GABAA receptors (H101R) escalated their binge-like drinking. Mutants harboring the Q241M point substitution in Gabra2 showed blunted chronic intake in the CA and IA protocols. S270H/L277A mutants consumed excessive amounts of alcohol but, unlike wild-types, they did not show forced abstinence-induced social deficits. CONCLUSIONS: These findings suggest a role for: (i) H101 in species-typical binge-like drinking, (ii) Q241 in escalated chronic drinking, and (iii) S270 and/or L277 in the development of forced abstinence-associated social deficits. Clinical findings report reduced BZD-binding sites in the cortex of dependent patients; the present findings suggest a specific role for BZD-sensitive α2-containing receptors. In addition, amino acid residue 241 in Gabra2 is necessary for positive modulation and activation of GABAA receptors by ALLO and THDOC; we postulate that neurosteroid action on α2-containing receptor may be necessary for escalated chronic EtOH intake.

Azogabazine; a photochromic antagonist of the GABAA receptor.

The design and synthesis of azogabazine is described, which represents a highly potent (IC50 = 23 nM) photoswitchable antagonist of the GABAA receptor. An azologization strategy is adopted, in which a benzyl phenyl ether in a high affinity gabazine analogue is replaced by an azobenzene, with resultant retention of antagonist potency. We show that cycling from blue to UV light, switching between trans and cis isomeric forms, leads to photochemically controlled antagonism of the GABA ion channel.

Disrupting the clustering of GABAA receptor α2 subunits in the frontal cortex leads to reduced γ-power and cognitive deficits.

In schizophrenia, cognitive dysfunction is highly predictive of poor patient outcomes and is not responsive to current medications. Postmortem studies have suggested that cognitive deficits in schizophrenia are correlated with modifications in the number and size of inhibitory synapses. To test if these modifications lead to cognitive deficits, we have created a dominant-negative virus [adeno-associated (AAV)-DN1] that disrupts the clustering of γ-aminobutyric acid type A receptors (GABA(A)Rs) at postsynaptic inhibitory specializations. When injected into the frontal cortex of mice, AAV-DN1 impairs GABA(A)R α2 subunit and GABA transporter 1 (GAT-1) clustering, but increases GABA(A)R α1 subunit clustering on the perisomatic region, with no influence on axon-initial segment clustering. Mice expressing AAV-DN1 have prepulse inhibition deficits and impairments in working memory. Significantly, these behavioral deficits are paralleled by a reduction in electroencephalography γ-power. Collectively, our study provides functional evidence revealing that GABAergic synapses in the prefrontal cortex directly contribute to cognition and γ-power.

Pharmacological characterisation of murine α4ß1δ GABAA receptors expressed in Xenopus oocytes.

BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor ß subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasynaptic GABAA receptor function is less well understood. Here, we examine the pharmacological properties of a potentially native extrasynaptic GABAA receptor that incorporates the ß1 subunit, specifically composed of α4ß1δ and α4ß1 subunits. RESULTS: GABA activated concentration-dependent responses at α4ß1δ and α4ß1 receptors with EC50 values in the nanomolar to micromolar range, respectively. The divalent cations Zn(2+) and Cu(2+), and the ß1-selective inhibitor salicylidine salicylhydrazide (SCS), inhibited GABA-activated currents at α4ß1δ receptors. Surprisingly the α4ß1 receptor demonstrated biphasic sensitivity to Zn(2+) inhibition that may reflect variable subunit stoichiometries with differing sensitivity to Zn(2+). The neurosteroid tetrahydro-deoxycorticosterone (THDOC) significantly increased GABA-initiated responses in concentrations above 30 nM for α4ß1δ receptors. CONCLUSIONS: With this study we report the first pharmacological characterisation of various GABAA receptor ligands acting at murine α4ß1δ GABAA receptors, thereby improving our understanding of the molecular pharmacology of this receptor isoform. This study highlights some notable differences in the pharmacology of murine and human α4ß1δ receptors. We consider the likelihood that the α4ß1δ receptor may play a role as an extrasynaptic GABAA receptor in the nervous system.

Sushi domains confer distinct trafficking profiles on GABAB receptors.

GABA(B) receptors mediate slow inhibitory neurotransmission in the brain and feature during excitatory synaptic plasticity, as well as various neurological conditions. These receptors are obligate heterodimers composed of GABA(B)R1 and R2 subunits. The two predominant R1 isoforms differ by the presence of two complement control protein modules or Sushi domains (SDs) in the N terminus of R1a. By using live imaging, with an α-bungarotoxin-binding site (BBS) and fluorophore-linked bungarotoxin, we studied how R2 stabilizes R1b subunits at the cell surface. Heterodimerization with R2 reduced the rate of internalization of R1b, compared with R1b homomers. However, R1aR2 heteromers exhibited increased cell surface stability compared with R1bR2 receptors in hippocampal neurons, suggesting that for receptors containing the R1a subunit, the SDs play an additional role in the surface stability of GABA(B) receptors. Both SDs were necessary to increase the stability of R1aR2 because single deletions caused the receptors to be internalized at the same rate and extent as R1bR2 receptors. Consistent with these findings, a chimera formed from the metabotropic glutamate receptor (mGluR)2 and the SDs from R1a increased the surface stability of mGluR2. These results suggest a role for SDs in stabilizing cell surface receptors that could impart different pre- and postsynaptic trafficking itineraries on GABA(B) receptors, thereby contributing to their physiological and pathological roles.

Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease.

Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) alpha1 subunit (GLRA1). Genetic heterogeneity has been confirmed in rare sporadic cases, with mutations affecting other postsynaptic glycinergic proteins including the GlyR beta subunit (GLRB), gephyrin (GPHN) and RhoGEF collybistin (ARHGEF9). However, many individuals diagnosed with sporadic hyperekplexia do not carry mutations in these genes. Here we show that missense, nonsense and frameshift mutations in SLC6A5 (ref. 8), encoding the presynaptic glycine transporter 2 (GlyT2), also cause hyperekplexia. Individuals with mutations in SLC6A5 present with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnea episodes. SLC6A5 mutations result in defective subcellular GlyT2 localization, decreased glycine uptake or both, with selected mutations affecting predicted glycine and Na+ binding sites.

Tyrosine phosphorylation of GABAA receptor γ2-subunit regulates tonic and phasic inhibition in the thalamus.

GABA-mediated tonic and phasic inhibition of thalamic relay neurons of the dorsal lateral geniculate nucleus (dLGN) was studied after ablating tyrosine (Y) phosphorylation of receptor γ2-subunits. As phosphorylation of γ2 Y365 and Y367 reduces receptor internalization, to understand their importance for inhibition we created a knock-in mouse in which these residues are replaced by phenylalanines. On comparing wild-type (WT) and γ2(Y365/367F)+/- (HT) animals (homozygotes are not viable in utero), the expression levels of GABAA receptor α4-subunits were increased in the thalamus of female, but not male mice. Raised δ-subunit expression levels were also observed in female γ2(Y365/367F) +/- thalamus. Electrophysiological analyses revealed no difference in the level of inhibition in male WT and HT dLGN, while both the spontaneous inhibitory postsynaptic activity and the tonic current were significantly augmented in female HT relay cells. The sensitivity of tonic currents to the δ-subunit superagonist THIP, and the blocker Zn(2+), were higher in female HT relay cells. This is consistent with upregulation of extrasynaptic GABAA receptors containing α4- and δ-subunits to enhance tonic inhibition. In contrast, the sensitivity of GABAA receptors mediating inhibition in the female γ2(Y356/367F) +/- to neurosteroids was markedly reduced compared with WT. We conclude that disrupting tyrosine phosphorylation of the γ2-subunit activates a sex-specific increase in tonic inhibition, and this most likely reflects a genomic-based compensation mechanism for the reduced neurosteroid sensitivity of inhibition measured in female HT relay neurons.

The major central endocannabinoid directly acts at GABA(A) receptors.

GABA(A) receptors are the major ionotropic inhibitory neurotransmitter receptors. The endocannabinoid system is a lipid signaling network that modulates different brain functions. Here we show a direct molecular interaction between the two systems. The endocannabinoid 2-arachidonoyl glycerol (2-AG) potentiates GABA(A) receptors at low concentrations of GABA. Two residues of the receptor located in the transmembrane segment M4 of ß(2) confer 2-AG binding. 2-AG acts in a superadditive fashion with the neurosteroid 3α, 21-dihydroxy-5α-pregnan-20-one (THDOC) and modulates δ-subunit-containing receptors, known to be located extrasynaptically and to respond to neurosteroids. 2-AG inhibits motility in CB(1)/CB(2) cannabinoid receptor double-KO, whereas ß(2)-KO mice show hypermotility. The identification of a functional binding site for 2-AG in the GABA(A) receptor may have far-reaching consequences for the study of locomotion and sedation.

Forty Years Searching for Neurosteroid Binding Sites on GABAA Receptors.

Brain inhibition is a vital process for controlling and sculpting the excitability of the central nervous system in healthy individuals. This level of control is provided over several timescales and involves the neurotransmitter GABA acting at inhibitory synapses to: rapidly inhibit neurons by activating the GABAA receptor; over a slower timescale, to tonically activate extrasynaptic GABAA receptors to provide a low level of background inhibition; and finally, to activate G-protein coupled GABAB receptors to control transmitter release by inhibiting presynaptic Ca2+ channels whilst providing postsynaptic inhibition via K+ channel activation. From this plethora of roles for GABA and its receptors, the GABAA receptor isoform is of major interest due to its dynamic functional plasticity, which in part, is due to being targeted by modulatory brain neurosteroids derived from sex and stress hormones. This family of neurosteroids can, depending on their structure, potentiate, activate and also inhibit the activity of GABAA receptors to affect brain inhibition. This review tracks the methods that have been deployed in probing GABAA receptors, and charts the sterling efforts made by several groups to locate the key neurosteroid binding sites that affect these important receptors. Increasing our knowledge of these binding sites will greatly facilitate our understanding of the physiological roles of neurosteroids and will help to advance their use as novel therapeutics to combat debilitating brain diseases.

Benzodiazepines modulate GABAA receptors by regulating the preactivation step after GABA binding.

GABA(A) receptors (GABA(A)Rs) composed of αßγ subunits are allosterically modulated by the benzodiazepines (BDZs). Agonists at the BDZ binding site potentiate submaximal GABA responses by increasing the apparent affinity of GABA(A)Rs for GABA. Although BDZs were initially thought to affect the binding of GABA agonists, recent studies suggest an effect on receptor gating; however, the involvement of preactivation steps in the modulation by BDZs has not been considered. Consequently, we examined whether BDZ agonists could exert their modulatory effect by displacing the equilibrium between resting and preactivated states of recombinant α1ß2γ2 GABA(A)Rs expressed in Xenopus oocytes. For GABA and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and piperidine-4-sulfonic acid, we examined BDZ modulation using a simple three-step model incorporating agonist binding, receptor preactivation, and channel opening. The model accounted for diazepam modulation simply by increasing the preactivation constant by approximately fourfold. To assess whether BDZs preferentially affected a specific GABA binding site, pentameric concatamers were used. This demonstrated that single GABA-binding site mutant receptors were equally sensitive to modulation by BDZs compared with wild-type counterparts. Overall, our results suggest that BDZs affect the preactivation step to cause a global conformational rearrangement of GABA(A)Rs, thereby modulating receptor function.

Protein kinase C regulates tonic GABA(A) receptor-mediated inhibition in the hippocampus and thalamus.

Tonic inhibition mediated by extrasynaptic GABA(A) receptors (GABA(A) Rs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA(A) Rs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA(A) R-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA(A) R activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4ß2δ GABA(A) Rs, which represent a key extrasynaptic GABA(A) R isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA(A) Rs. The inhibitory effects of PKC activation on α4ß2δ GABA(A) R activity appeared to be mediated by direct phosphorylation at a previously identified site on the ß2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABA(A) R-mediated inhibition.

Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.

Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.