RESUMO
γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the adult brain which mediates its rapid effects on neuronal excitability via ionotropic GABAA 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. OV329 is a rationally designed, next-generation GABA-AT inhibitor with enhanced potency. We demonstrate that sustained exposure to OV329 in mice reduces GABA-AT activity and subsequently elevates GABA levels in the brain. Parallel increases in the efficacy of GABAergic inhibition were evident, together with elevations in electroencephalographic delta power. Consistent with this, OV329 exposure reduced the severity of status epilepticus and the development of benzodiazepine refractory seizures. Thus, OV329 may be of utility in treating seizure disorders and associated pathologies that result from neuronal hyperexcitability.
Assuntos
4-Aminobutirato Transaminase , Anticonvulsivantes , Benzodiazepinas , Convulsões , Ácido gama-Aminobutírico , Animais , Anticonvulsivantes/farmacologia , Anticonvulsivantes/administração & dosagem , Masculino , Benzodiazepinas/farmacologia , 4-Aminobutirato Transaminase/antagonistas & inibidores , 4-Aminobutirato Transaminase/metabolismo , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Ácido gama-Aminobutírico/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Camundongos , Eletroencefalografia , Modelos Animais de Doenças , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , FemininoRESUMO
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. KCC2 activity is essential for Cl--mediated hyperpolarizing GABAAR receptor currents, the unitary events that underpin fast synaptic inhibition. LMTK3 acts to promote the association of KCC2 with PP1 to promote the dephosphorylation of S940 within its C-terminal cytoplasmic domain, a process the diminishes KCC2 activity. Accordingly, acute inhibition of LMTK3 increases KCC2 activity dependent upon S940 and increases neuronal Cl- extrusion. Consistent with this, LMTK3 inhibition reduced intrinsic neuronal excitability and the severity of seizure-like events in vitro. Thus, LMTK3 may have profound effects on neuronal excitability as an endogenous modulator of KCC2 activity.
RESUMO
Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, ß and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. ß2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while ß4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating ß2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition.
Assuntos
Receptores de GABA-A , Espectrina , Receptores de GABA-A/metabolismo , Espectrina/metabolismo , Sinapses/metabolismo , Proteínas de Membrana/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
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 γ-aminobutyric acid type A receptors (GABA A Rs), which mediate phasic and tonic inhibition, respectively. These two GABA A R subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABA A Rs contain the α1-3 subunits whereas extrasynaptic GABA A Rs contain the α4-6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABA A R subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABA A Rs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABA A Rs and interact with distinct sets of binding proteins. We also discovered that the ß3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABA A Rs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABA A Rs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABA A Rs, and thus the efficacy of neuronal inhibition.
RESUMO
Neuroactive steroids (NASs) have potent anxiolytic, anticonvulsant, sedative, and hypnotic actions, that reflect in part their efficacy as GABA A R positive allosteric modulators (PAM). In addition to this, NAS exert metabotropic effects on GABAergic inhibition via 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 ß3 subunit of GABA A Rs, increasing their accumulation in the plasma membrane leading to a sustained increase in tonic inhibition. To explore the significance of NAS-induced phosphorylation of GABA A Rs, we used mice in which S408/9 in the ß3 subunit have been mutated to alanines, mutations that prevent the metabotropic actions of NASs on GABA A R function while preserving NAS allosteric potentiation of GABAergic current. While the sedative actions of NAS were comparable to WT, their anxiolytic actions were reduced in S408/9A mice. Although the induction of hypnosis by NAS were maintained in the mutant mice the duration of the loss of righting reflex was significantly shortened. Finally, ability of NAS to terminate diazepam pharmacoresistant seizures was abolished in S408/9A mice. In conclusion, our results suggest that S408/9 in the GABA A R ß3 subunit contribute to the anxiolytic and anticonvulsant efficacy of NAS, in addition to their ability to regulate the loss of righting reflex.
RESUMO
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-HT3A serotonin or α1 glycine receptors, serotonin and Zn2+/H+ evoked robust concentration-dependent currents in 5-HT3A/ZAC- and ZAC/α1-Gly-expressing oocytes, respectively, suggesting that Zn2+ and protons activate ZAC predominantly through its ECD. The molecular basis for Zn2+-mediated ZAC signaling was probed further by introduction of mutations of His, Cys, Glu and Asp residues in this domain, but as none of the mutants tested displayed substantially impaired Zn2+ functionality compared to wild-type ZAC, the location of the putative Zn2+ binding site(s) in the ECD was not identified. Finally, the functional importance of Leu246 (Leu9') in the transmembrane M2 α-helix of ZAC was investigated by Ala, Val, Ile and Thr substitutions. In concordance with findings for this highly conserved residue in classical CLRs, the ZACL9'X mutants exhibited left-shifted agonist concentration-response relationships, markedly higher degrees of spontaneous activity and slower desensitization kinetics compared to wild-type ZAC. In conclusion, while ZAC is an atypical CLR in terms of its (identified) agonists and channel characteristics, its signal transduction seems to undergo similar conformational transitions as those in the classical CLR.
Assuntos
Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Animais , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Proteínas do Tecido Nervoso/genética , Oócitos , Subunidades Proteicas , Proteínas Recombinantes de Fusão , Xenopus , Zinco/farmacologiaRESUMO
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 (IC50 values: 1-3 µM). 1 and N-(4-(tert-butyl)thiazol-2-yl)-3-fluorobenzamide (5a, TTFB) were next applied in studies of the functional properties and the mode of action of this novel class of ZAC antagonists. TTFB was a roughly equipotent antagonist of Zn+- and H+-evoked ZAC signaling and of spontaneous ZAC activity, and the slow on-set of its channel block suggested that its ZAC inhibition is state-dependent. TTFB was found to be a selective ZAC antagonist, exhibiting no significant agonist, antagonist or modulatory activity at 5-HT3A, α3ß4 nicotinic acetylcholine, α1ß2γ2S GABAA or α1 glycine receptors at 30 µM. 1 displayed largely non-competitive antagonism of Zn2+-induced ZAC signalling, and TTFB was demonstrated to target the transmembrane and/or intracellular domains of the receptor, which collectively suggests that the N-(thiazol-2-yl)-benzamide analog acts a negative allosteric modulator of ZAC. We propose that this first class of selective ZAC antagonists could constitute useful pharmacological tools in future explorations of the presently poorly elucidated physiological functions governed by this CLR.
Assuntos
Benzamidas/farmacologia , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/antagonistas & inibidores , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Animais , Benzamidas/química , Receptores de Canais Iônicos de Abertura Ativada por Ligante com Alça de Cisteína/genética , Descoberta de Drogas , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/genética , Oócitos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , XenopusRESUMO
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 Thr128Ala variation in ZAC is found in extremely high allele frequencies across ethnicities. In this, the first study of ZAC in Xenopus oocytes by TEVC electrophysiology, ZACThr128 and ZACAla128 exhibited largely comparable pharmacological and signalling characteristics, but interestingly the Zn2+- and H+-evoked current amplitudes in ZACAla128-oocytes were dramatically smaller than those in ZACThr128-oocytes. While the variation thus appeared to impact cell surface expression and/or channel properties of ZAC, the similar expression properties exhibited by ZACThr128 and ZACAla128 in transfected mammalian cells indicated that their distinct functionalities could arise from the latter. In co-expression experiments, wild-type and variant ZAC subunits assembled efficiently into "heteromeric" complexes in HEK293 cells, while the concomitant presence of ZACAla128 in ZACThr128:ZACAla128-oocytes did not exert a dominant negative effect on agonist-evoked current amplitudes compared to those in ZACThr128-oocytes. Finally, the structural determinants of the functional importance of the 1-hydroxyethyl side-chain of Thr128 appeared to be subtle, as agonist-evoked current amplitudes in ZACSer128-, ZACVal128- and ZACIle128-oocytes also were substantially lower than those in ZACThr128-oocytes. In conclusion, the functional properties exhibited by ZAC in this work substantiate the notion of it being an atypical CLR. While the impact of the Thr128Ala variation on ZAC functionality in oocytes is striking, it remains to be investigated whether and to which extent this translates into an in vivo setting and thus could constitute a source of inter-individual variation in ZAC physiology.
Assuntos
Canais Iônicos/metabolismo , Zinco/metabolismo , Animais , Ensaio de Imunoadsorção Enzimática , Imunoprecipitação , Canais Iônicos/genética , Canais Iônicos/fisiologia , Oócitos/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Xenopus laevisRESUMO
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 (GABAARs). 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 GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.
Assuntos
Neurônios/metabolismo , Simportadores/metabolismo , Animais , Apoptose , Cloretos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Cultura Primária de Células , Receptores de GABA/metabolismo , Convulsões , Simportadores/fisiologia , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
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. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.
RESUMO
Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1ß2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram ß-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.
Assuntos
Anticonvulsivantes/farmacologia , Moduladores GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Pregnanos/farmacologia , Pirazóis/farmacologia , Esteroides/farmacologia , Animais , Anticonvulsivantes/farmacocinética , Antidepressivos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Diazepam/farmacologia , Sinergismo Farmacológico , Eletroencefalografia/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pregnanos/farmacocinética , Pirazóis/farmacocinética , Ratos Sprague-Dawley , Receptores de GABA/efeitos dos fármacos , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Ácido gama-Aminobutírico/fisiologiaRESUMO
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 (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype-the α2-containing receptor-is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.
RESUMO
The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABAARs) at the appropriate synaptic sites. Synaptic GABAARs are constructed from α(1-3), ß(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 GABAARs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments.
Assuntos
Motivos de Aminoácidos/genética , Axônios/metabolismo , Neurônios GABAérgicos/metabolismo , Receptores de GABA-A/metabolismo , Convulsões/metabolismo , Sinapses/metabolismo , Animais , Axônios/fisiologia , Células Cultivadas , Neurônios GABAérgicos/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores de GABA-A/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Convulsões/genética , Convulsões/mortalidade , Sinapses/genética , Transmissão Sináptica/fisiologiaRESUMO
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 (GABAAR). NASs also activate a family of G protein-coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABAAR ß3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABAAR activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the ß3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed "NAS antagonist," the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABAAR phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.
Assuntos
Neuroesteroides/metabolismo , Receptores de GABA-A/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Membrana Celular/metabolismo , Potenciais Evocados/efeitos dos fármacos , Antagonistas de Receptores de GABA-A/farmacologia , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Neuroesteroides/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/genética , Receptores de Progesterona/agonistas , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
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 (GABAARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAAR 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 ß3 subunits which increase the membrane expression and boosts tonic inhibition. We have assessed the GABAergic signaling in the hippocampus of fragile X mental retardation protein (FMRP) knock-out (Fmr1 KO) mouse. The GABAergic tonic current in dentate gyrus granule cells (DGGCs) from 3- to 5-week-old (p21-35) Fmr1 KO mice was significantly reduced compared to WT mice. Additionally, spontaneous inhibitory post synaptic inhibitory current (sIPSC) amplitudes were increased in DGGCs from Fmr1 KO mice. While sIPSCs decay in both genotypes was prolonged by the prototypic benzodiazepine diazepam, those in Frm1-KO mice were selectively potentiated by RO15-4513. Consistent with this altered pharmacology, modifications in the expression levels and phosphorylation of receptor GABAAR subtypes that mediate tonic inhibition were seen in Fmr1 KO mice. Significantly, exposure to NASs induced a sustained elevation in tonic current in Fmr1 KO mice which was prevented with PKC inhibition. Likewise, exposure reduced elevated membrane excitability seen in the mutant mice. Collectively, our results suggest that NAS act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability seen in this disorder.
Assuntos
Receptores de GABA-A/efeitos dos fármacos , Simportadores/efeitos dos fármacos , Tiazolidinas/farmacologia , Linhagem Celular Tumoral , Cloretos/metabolismo , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Ligação Proteica , Simportadores/metabolismo , Tálio/farmacologia , Transfecção , Cotransportadores de K e Cl-RESUMO
The neuroactive steroid (NAS) tetrahydrodeoxycorticosterone (THDOC) increases protein kinase C (PKC) mediated phosphorylation of extrasynaptic GABAA receptor (GABAAR) subunits leading to increased surface expression of α4/ß3 subunit-containing extrasynaptic GABAARs, leading to a sustained increase in GABAAR tonic current density. Whether other naturally occurring and synthetic NASs share both an allosteric and metabotropic action on GABAARs is unknown. Here, we examine the allosteric and metabotropic properties of allopregnanolone (ALLO), and synthetic NASs SGE-516 and ganaxolone. ALLO, SGE-516, and ganaxolone all allosterically enhanced prototypical synaptic and extrasynaptic recombinant GABAARs. In dentate gyrus granule cells (DGGCs) all three NASs, when applied acutely, allosterically enhanced tonic and phasic GABAergic currents. In separate experiments, slices were exposed to NASs for 15 min, and then transferred to a steroid naïve recording chamber followed by ≥ 30 min wash before tonic currents were measured. A sustained increase in tonic current was observed following exposure to ALLO, or SGE-516 and was prevented by inhibiting PKC with GF 109203X. No increase in tonic current was observed with exposure to ganaxolone. In agreement with the observations of an increased tonic current, the NASs ALLO and SGE-516 increased the phosphorylation and surface expression of the ß3 subunit-containing GABAARs. Our studies demonstrate that neuroactive steroids have differential abilities to induce sustained increases in the efficacy of tonic inhibition by promoting GABAAR phosphorylation and membrane trafficking dependent on PKC activity.
Assuntos
Desoxicorticosterona/análogos & derivados , Inibição Neural/fisiologia , Pregnanolona/farmacologia , Proteína Quinase C/metabolismo , Receptores de GABA-A/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Desoxicorticosterona/farmacologia , Inibidores Enzimáticos/farmacologia , Células HEK293 , Hipocampo , Humanos , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Inibição Neural/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Proteína Quinase C/antagonistas & inibidoresRESUMO
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. GABAergic signaling allows for precise synchronized firing of action potentials within brain circuits that is critical for cognition, behavior, and consciousness. This precision depends upon tightly controlled trafficking of GABAARs into the membrane. General anesthetics bind to and allosterically enhance GABAARs by prolonging the open state of the receptor and thereby altering neuronal and brain circuit activity. Subunit composition and GABAAR localization strongly influence anesthetic end points; therefore, changes in GABAAR trafficking could have significant consequences to anesthetic sensitivity. GABAARs are not static membrane structures but are in a constant state of flux between extrasynaptic and synaptic locations and are continually endocytosed and recycled from and to the membrane. Neuronal activity, posttranslational modifications, and some naturally occurring and synthetic compounds can influence the expression and trafficking of GABAARs. In this article, we review GABAARs, their trafficking, and how phosphorylation of GABAAR subunits can influence the surface expression and function of the receptor. Ultimately, alterations of GABAAR trafficking could modify anesthetic end points, both unintentionally through pathologic processes but potentially as a therapeutic target to adjust anesthetic-sensitive GABAARs.
Assuntos
Anestésicos Gerais/metabolismo , Antagonistas GABAérgicos/metabolismo , Inibição Neural/fisiologia , Receptores de GABA-A/metabolismo , Anestésicos Gerais/farmacologia , Animais , Antagonistas GABAérgicos/farmacologia , Humanos , Inibição Neural/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
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. In this study, we sought to understand how decreased KCC2 transport function affects seizure event severity. We impaired KCC2 transport in the 0-Mg(2+) ACSF and 4-aminopyridine in vitro models of epileptiform activity in acute mouse brain slices. Experiments with the selective KCC2 inhibitor VU0463271 demonstrated that reduced KCC2 transport increased the duration of SLEs, resulting in non-terminating discharges of clonic-like activity. We also investigated slices obtained from the KCC2-Ser940Ala (S940A) point-mutant mouse, which has a mutation at a known functional phosphorylation site causing behavioral and cellular deficits under hyperexcitable conditions. We recorded from the entorhinal cortex of S940A mouse brain slices in both 0-Mg(2+) ACSF and 4-aminopyridine, and demonstrated that loss of the S940 residue increased the susceptibility of continuous clonic-like discharges, an in vitro form of status epilepticus. Our experiments revealed KCC2 transport activity is a critical factor in seizure event duration and mechanisms of termination. Our results highlight the need for therapeutic strategies that potentiate KCC2 transport function in order to decrease seizure event severity and prevent the development of status epilepticus.
Assuntos
Convulsões/induzido quimicamente , Convulsões/metabolismo , Simportadores/metabolismo , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Simportadores/antagonistas & inibidores , Cotransportadores de K e Cl-RESUMO
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, ß, 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). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.